Chronic Endothelin-A Receptor Blockade

in Lambs with Increased Pulmonary Blood Flow and Pressure

Sohrab Fratz1,2, Barbara Meyrick3, Boaz Ovadia2, Michael J. Johengen2, Olaf Reinhartz2,

Anthony Azakie2, Greg Ross2, Robert Fitzgerald2, Peter Oishi2, John Hess1, Stephen M. Black4,

Jeffrey R. Fineman2

1) Department of Pediatric Cardiology and Congenital Heart Disease, Deutsches Herzzentrum

München, Munich, Germany

2) Department of Pediatrics and the Cardiovascular Research Institute, University of California, San

Francisco, CA, USA

3) Departments of Pathology and Medicine Vanderbilt University, Nashville, TN, USA

4) Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, MT,

USA.

Running head: ETA block and increased pulmonary blood flow

Contact information: Sohrab Fratz, MD, Dept. of Pediatric Cardiology and Congenital Heart Disease,

Deutsches Herzzentrum München, Klinik an der Technischen Universität München, Lazarettstr. 36,

80636 Munich, Germany. E-mail: fratz@dhm.mhn.de; Phone: +49 89 1218 3031; Fax: +49 89 1218 3013.

Articles in PresS. Am J Physiol Lung Cell Mol Physiol (May 21, 2004). 10.1152/ajplung.00093.2004

Copyright © 2004 by the American Physiological Society.

ETA block and increased pulmonary blood flow 1

ABSTRACT

Endothelin-receptor blockade is an emerging therapy for pulmonary hypertension. However,

hemodynamic and structural effects and potential changes in endogenous NO-cGMP and endothelin-1

signalling of chronic endothelinA-receptor blockade in pulmonary hypertension secondary to congenital

heart disease are unknown. Therefore the objectives of this study were to determine hemodynamic and

structural effects and potential changes in endogenous NO-cGMP and endothelin-1 signalling of chronic

endothelinA-receptor blockade in a lamb model of increased pulmonary blood flow following in-utero

placement of an aortopulmonary shunt. Immediately after spontaneous birth shunt-lambs were treated

lifelong with either an endothelinA-receptor antagonist (PD156707) or placebo. At four weeks age,

PD156707-treated shunt-lambs (n=6) had lower pulmonary vascular resistance and right atrial pressure

than placebo-treated shunt-lambs (n=8) (p<0.05). Smooth muscle thickness or arterial number per unit

area was not different between the two groups. However, the number of alveolar profiles per unit area

was increased in the PD156707-treated shunt-lambs (190.7±5.6 vs. 132.9±10.0, p<0.05). Plasma

endothelin-1 and cGMP levels, and lung NOS activity, cGMP, eNOS, preproendothelin-1, endothelin-

converting-enzyme-1, endothelinA and endothelinB-receptors protein levels were similar in both groups.

We conclude that chronic endothelinA-receptor blockade attenuates the progression of pulmonary

hypertension and augments alveolar growth in lambs with increased pulmonary blood flow.

Key Words: Endothelin; Pulmonary Heart Disease; Congenital Heart Defect

ETA block and increased pulmonary blood flow 2

INTRODUCTION

Altered pulmonary vascular reactivity and pulmonary hypertension is a common accompaniment

of congenital heart disease with increased pulmonary blood flow1. Normal pulmonary vascular tone and

vascular smooth muscle cell proliferation is regulated by a complex interaction of vasoactive substances

that are locally produced by the vascular endothelium, such as nitric oxide (NO) and endothelin-1 (ET-

1)2-4. Endothelial injury secondary to increased pulmonary blood flow and/or pressure disrupts these

regulatory mechanisms, and is a potential factor in the development of pulmonary hypertension secondary

to congenital heart disease5,6.

ET-1 is a 21 amino acid polypeptide produced by vascular endothelial cells that has potent

vasoactive properties, and is co-mitogenic for vascular smooth muscle cells4,7. Recent data suggest a role

for ET-1 in the pathophysiology of pulmonary hypertension. For example, ET-1 concentrations are

increased in children with increased pulmonary blood flow and pulmonary hypertension, and preproET-1

gene expression is increased in adults with advanced pulmonary vascular disease6,8,9. In addition, chronic

endothelin receptor blockade has demonstrated clinical improvement in adults with advanced pulmonary

vascular disease10. However, the early role of ET-1 in the pathophysiology of pulmonary hypertension

secondary to congenital heart disease with increased pulmonary blood flow remains unclear11. In addition,

the potential effects of chronic ET receptor blockade on endogenous endothelial signaling are unknown.

To better define the role of ET-1 in the pathogenesis of pulmonary hypertension, we established a

model of pulmonary hypertension with increased pulmonary blood flow in the lamb, following in utero

placement of an aorta-to-pulmonary vascular graft. At one month of age, these lambs (shunt-lambs) have

a pulmonary to systemic blood flow ratio of approximately 2:1, a mean pulmonary arterial pressure that is

50% of mean systemic arterial pressure, and pulmonary vascular remodeling characteristic of children

ETA block and increased pulmonary blood flow 3

with pulmonary hypertension and increased pulmonary blood flow12. Previously, we demonstrated that

shunt lambs display alterations in the ET-1 cascade at 4 weeks of age. These include increased plasma

ET-1 levels, decreased ETB receptor protein with loss of ETB receptor-mediated vasodilation, and

increased ETA receptor protein with augmentation of ETA receptor-mediated vasoconstriction, suggesting

a role for ET-1 in the pathophysiology of pulmonary hypertension secondary to increased pulmonary

blood flow13,14.

The objectives of this study were to determine the role of ETA receptor activity in the early

progression of pulmonary hypertension secondary to congenital heart disease with increased pulmonary

blood flow, and determine the potential effects of ET receptor blockade on endothelial signaling.

Therefore, we studied the effects of chronically administered PD156707 (an ETA receptor antagonist), or

placebo, in our lamb model of congenital heart disease. Four weeks after spontaneous delivery of the

lambs, hemodynamic variables, ET-1-induced vasoactive responses, and pulmonary artery morphology

were determined and compared. In addition, potential alterations in the endogenous NO-cGMP and ET-1

pathways induced by chronic ETA receptor blockade were determined by comparing plasma ET-1, plasma

and lung tissue NOx and cGMP levels, cGMP levels, tissue nitric oxide synthase [NOS] activity, and

tissue endothelial NOS, preproET-1, ECE-1, and ETA and ETB receptor protein levels in lambs with and

without ET receptor blockade.

ETA block and increased pulmonary blood flow 4

METHODS

Surgical Preparation and Care

Ewes

Twenty mixed-breed Western pregnant ewes (135-141 days gestation, term = 145 days) were operated on

as previously described12-14.

Lambs

Before spontaneous delivery, the lambs were randomly divided into a treatment and a placebo

group. The treatment group (n=12) received PD156707, an ETA receptor antagonist diluted in water (150

mg/kg/d). The placebo group (n=8) received daily water. All treatments were started within 12 hours of

spontaneous delivery, and continued for 4 weeks. The lambs were weighed daily and the respiratory rate

and heart rates were obtained weekly by a animal care nurse who was blinded to the therapy. Furosemide

(1 mg/kg IM) was administered twice daily. At 4 weeks of age, vascular pressures and flow was measured

as previously described13,14.

Experimental Protocol

After baseline measurements of the hemodynamic variables were obtained, ET-1 (250 µg/kg) was

injected into the pulmonary artery over 1 minute. The hemodynamic variables were measured

continuously.

ETA block and increased pulmonary blood flow 5

Tissue preparation

Blocks of tissue of the left lung were removed and stored for later Western Blot analysis.

Additionally, in five of the PD156707-treated shunt-lambs and five of the placebo-treated shunt-lambs,

the pulmonary arterial bed of the right lung was distended with a barium gelatin suspension and the lungs

were inflated by way of the trachea with formalin and placed in a bath of formalin for fixation.

Six random blocks for routine light microscopic examination were taken from each left lung. The

sections were then examined for the characteristic structural changes of chronic pulmonary hypertension

by the use of well-established quantitative techniques15,16. Briefly, external diameter of at least 100 arterial

profiles was measured as well as medial thickness of the muscular and partially muscular arteries. Medial

thickness was then related to arterial size using the calculation: percent medial thickness = 2 X medial

thickness/external diameter X 100. The structure of each artery was also noted -- muscular, partially

muscular, nonmuscular -- as was the structure of the accompanying airway -- bronchus, bronchiolus,

terminal bronchiolus, respiratory bronchiolus, alveolar duct, and alveolar wall. The density of the barium

filled intraacinar arteries was also assessed. Using a X 25 objective and an eyepiece reticule, the number

of barium filled arteries of less than 200 µm external diameter was counted and related to the number of

alveolar profiles in these same fields. Lastly, the number of alveolar per unit area were counted. At least

25 consecutive microscopic fields were counted for each animal.

Plasma and Tissue Determinations

Blood samples were assayed for immunoreactive endothelin (irET-1) as previously described13.

Blood samples and tissue were assayed with a cGMP [125I] assay kit (Amersham International

plc, Amersham UK) according to manufacturers instructions17.

ETA block and increased pulmonary blood flow 6

Utilizing a vanadium (III) and hydrochloric acid reduction, total NO, nitrite, and nitrate (NOx)

were detected by chemiluminescence (NOA 280, Sievers Instruments Inc. Boulder CO) in blood samples

and lung tissue18.

NOS activity in the lung tissue was determined by using the conversion of 3H-arginine to 3H-

citrulline as previously described19.

Western Blot Analysis

Western blot analysis (ETA and ETB receptor, prepro-Endothelin-1, ECE-1, endothelial NOS) was

performed as previously described14.

Statistical Analysis

The mean ± standard deviation was calculated for the hemodynamic variables, systemic arterial

blood gases and pH and ET-1 levels, structural changes, cGMP, NOx, and NOS activity. Comparisons

were made by the paired t-test using the Bonferroni correction, the unpaired t-test or ANOVA for

repeated measures with multiple comparison testing.

Quantitation of autoradiographic results was performed by scanning the bands of interest into an

image editing software program (Adobe Photoshop, Adobe Systems, Mt. View, CA). The means ±

standard deviation was calculated for the relative protein. Protein and structural data were compared by

the unpaired t-test. A P < 0.05 was considered statistically significant.

ETA block and increased pulmonary blood flow 7

RESULTS

Spontaneous delivery occurred 2-19 days after fetal surgery. Six of the PD156707-treated shunt-

lambs died between day 1 and 21 of life. Two lambs died from presumed sepsis, one from necrotic

enterocolitis, and one sudden death from unknown causes. Two additional lambs were sacrificed because

of respiratory distress following an aspiration event. All of the placebo-treated shunt-lambs survived the 4-

week study period. In general, we observed that the lambs in the treatment group were clinically sicker,

with increased work of breathing and decreased activity level. However, this could not be quantified and

there were no differences in body weight, respiratory rate, or heart rate between the 2 groups (data not

shown). A total of 14 lambs (n=6 treated and n=8 placebo) completed the 4-week study period, and

underwent the hemodynamic study and biochemical and morphometric analysis.

Hemodynamic Study

All lambs had an audible continuous murmur and an increase in oxygen saturation between the

right ventricle and distal pulmonary artery. The baseline systemic arterial pCO2, pO2, and hemoglobin,

were similar between the two groups and within the normal limits of the laboratory. There were no

differences in systemic arterial pH between the two groups (7.46±0.04 vs. 7.52±0.06), however both were

greater than historical controls for the laboratory13,14. Left pulmonary vascular resistance, mean right atrial

pressure, and systemic pulse pressure were decreased in PD156707-treated shunt-lambs compared to

placebo-treated shunt-lambs (p<0.05, Table 1). All other hemodynamic variables measured were not

different between the two groups. However, mean pulmonary arterial pressure in PD156707-treated shunt-

lambs was similar to values previously noted in age-matched control lambs13,14.

ETA block and increased pulmonary blood flow 8

In placebo-treated shunt-lambs, the intrapulmonary injection of ET-1 increased left pulmonary

vascular resistance (18.5%±26.0) and mean systemic arterial pressure (9.4% ± 5.0) (p<0.05). However, in

all PD156707-treated shunt-lambs both the pulmonary and systemic vasoconstriction to ET-1 was blocked.

Biochemical and Protein Determinations

Lung tissue NOx levels, plasma and lung tissue cGMP levels, plasma ET-1 (P=0.06), and tissue

NOS activity were all similar between the two groups. Plasma NOx levels were decreased in PD156707-

treated shunt-lambs when compared to placebo-treated shunt-lambs (P<0.05, Table 2). Protein levels of

eNOS, preproET-1, ECE-1, ETA and ETB receptors were are similar between the two groups (Table 3).

Structural Studies

Morphometric analysis revealed no differences in the percent medial thickness of pulmonary

arteries between the two groups (Figure 1). However, the presence of muscular arteries appearance in the

intraacinar region at the alveolar duct and alveolar wall levels was significantly increased in the

PD156707-treated shunt-lambs compared to the placebo-treated shunt-lambs (Table 4). In addition, while

there was no difference in the number of barium-filled small arteries per unit area between the two groups

(3.8 ± 0.2, n = 5 vs. 4.0 ± 0.3, n = 5), the number of alveolar profiles per unit area was increased by 43.5%

in PD156707-treated shunt-lambs (p<0.05) (Figure 2). This led to a significant reduction in number of

small barium-filled arteries per 100 alveolar profiles in the PD156707-treated shunt-lambs (2.1 ± 0.3 vs.

2.9 ± 0.3 arteries / 100 alveoli, p = 0.002).

ETA block and increased pulmonary blood flow 9

DISCUSSION

Activation of the ET-1 cascade has been demonstrated in a variety of pulmonary hypertensive

disorders including children and animals with congenital heart disease and increased pulmonary blood

flow. The present study is the first to demonstrate that chronic endothelin receptor blockade lowers

pulmonary vascular resistance in a model of increased pulmonary blood flow secondary to congenital

heart disease, suggesting a role for ET-1 in the early pathophysiology of this disorder. Following in utero

aortopulmonary graft placement in the lamb, we found that chronic postnatal ETA receptor blockade

lowered pulmonary vascular resistance and right atrial pressure without affecting basal endogenous NO-

cGMP or ET-1 signalling. We also demonstrate the novel finding that ETA receptor blockade amplified

alveolar growth, suggesting a role for ET-1 in the regulation of postnatal alveolar development in this

setting.

Although chronic ET receptor blockade has demonstrated symptomatic improvement in adults

with advanced primary pulmonary hypertension, this would not be expected in the setting of increased

pulmonary blood flow10. Since it is well established that pulmonary vasodilator therapy in the setting of a

left-to-right shunt worsens congestive heart failure and mortality, the objective of this study was to utilize

PD156707 as a pharmacologic tool to further delineate the role of ET-1 in the pathophysiology of this

disorder, rather than a potential therapy in this setting20. As expected, PD156707-treated shunt-lambs had

increased work of breathing, decreased activity, and a greater mortality (50% vs. 0%, P<0.05) than

placebo-treated shunt-lambs. Postmortem investigation of the deaths suggested that two occurred from

aspiration events, one from an intrauterine infection, one from a postnatal urachus infection, and one

sudden death of unclear etiology. In an attempt to prevent the increased symptomatology of increased

pulmonary blood flow, both groups of lambs were treated with furosemide twice a day, beginning on the

ETA block and increased pulmonary blood flow 10

first day of life. It is noteworthy that both PD156707 and placebo-treated shunt-lambs had lower

pulmonary arterial pressure, higher systemic arterial pH, and less vascular remodelling than historical

shunt controls, which only received furosemide on an intermittent basis in response to increased

symptomatology12. The effects of the aggressive diuretic regimen, in addition to the high mortality of the

treatment group were unavoidable, but significant limitations of the current study. We cannot exclude the

possibility that the high mortality selected out lambs with differential hemodynamics and vascular

morphology, and that a shorter treatment course with a less aggressive diuretic regimen may have

demonstrated greater differences between the PD156707 and placebo-treated shunt-lambs. Additionally,

we also cannot exclude the possibility that the 50% mortality has introduced other unknown serious biases

to the study. Further studies are needed to better delineate these potential factors.

In previous animal models of pulmonary hypertension, chronic ET receptor blockade attenuated

the progression of vascular smooth muscle remodeling. For example in rats exposed to either hypoxia or

monocrotaline, ET receptor blockade resulted in decreased medial thickness of the small pulmonary

arteries compared to controls21,22. Similarly, in postnatal piglets with increased pulmonary blood flow, ET

receptor blockade prevented the increase in medial thickness of the small pulmonary arteries23. However,

ETA receptor blockade did not prevent pulmonary vascular remodelling in a rat model of myocardial

infarction24. In the present study, PD156707-treated shunt-lambs displayed no differences in percent

medial thickness of the pulmonary arteries compared to controls. In fact, the abnormal presence of

muscularized pulmonary arteries in the region of the alveolar duct and wall was greater in PD156707-

treated shunt-lambs (Table 4). These surprising findings are not completely understood, but a few

potential explanations are noteworthy. First, as previously mentioned, the degree of vascular remodeling

was not generally significant in neither the PD156707-treated shunt-lambs nor the placebo-treated shunt-

lambs, and much less than historical shunt controls, which is likely due to the aggressive diuretic regimen

ETA block and increased pulmonary blood flow 11

utilized in this study. Second, the high mortality in the PD156707-treated shunt-lambs may have selected

for a group of survivors with more vascular remodelling, which could limit pulmonary blood flow and

clinical symptomatology. Lastly and most importantly, this study is unique in that the abnormal

hemodynamic state was present from birth, and treatment was started immediately following birth. Since

the early morphologic abnormalities in children with increased pulmonary blood flow are similar to the

normal fetal vascular morphology, it is likely that the early vascular changes represent a failure of the

fetal morphologic state to regress rather that remodelling. Therefore, the abnormal extension of muscle

present in the PD156707-treated shunt-lambs may represent a failure of the normal regression of fetal

morphology, and may suggest a role for ET-1 in this process.

Previously, we demonstrated that increased pulmonary blood flow induces significant alterations

in the endogenous ET-1 and NO-cGMP cascades13,14,25. In the present study, we demonstrate that chronic

ETA receptor blockade does not significantly alter these changes. For example, there were no differences

in plasma ET-1 levels, plasma and tissue cGMP levels, NOS activity, and protein levels of eNOS,

preproET-1, ECE-1, ETA and ETB receptors. Plasma NOx levels were increased in PD156707-treated

shunt-lambs. However, plasma NOx levels may be altered by differences in extracellular volume and

renal clearance26. Since lung tissue NOx levels were unchanged, differences in plasma NOx are unlikely

to represent differences in basal pulmonary NO production.

To achieve ETA receptor blockade treated lambs received 150mg/kg/dy of PD156707, an orally

active, selective ETA receptor antagonist27. The dose of PD156707 was chosen after several previous

studies showed that this dose completely blocked the vasoconstricting effects of exogenous ET-1 and

resulted in steady-state plasma concentrations that blocked ETA receptors in vivo28. To insure adequate

ETA receptor blockade with PD156707 in this study, the effect of exogenous ET-1 was studied in both

groups of lambs. Similar to our previous studies ET-1 induced both pulmonary and systemic

ETA block and increased pulmonary blood flow 12

vasoconstriction in vehicle-treated lambs13. However, in all PD156707-treated lambs, the vasoconstricting

effect of ET-1 was blocked suggesting adequate ETA receptor blockade. Interestingly, ET-1 did not elicit

pulmonary vasodilation. These physiologic data in conjunction with the protein data demonstrating no

change in ETB receptor protein, suggest that ETB receptors were not up-regulated during ETA receptor

blockade.

Lastly, this study demonstrates the novel finding that chronic ETA receptor blockade increases

alveolar, and presumably capillary, growth in lambs with increased pulmonary blood flow. As seen in

Figure 2, the number of alveolar-capillary profiles per unit area was increased by 44% in PD156707-

treated shunt-lambs (P<0.05). Although ET-1 is known to have mitogenic actions on cardiovascular

tissues, and is co-mitogenic with a variety of growth factors and vasoactive substances, there is very

limited data on the role of ET-1 in the developing lung29. However, ET-1 is expressed in the airway

epithelium of the perinatal mouse, suggesting a role for ET-1 in lung development30. In the current study,

we have uncovered a previously unknown role for ET-1 in postnatal lung development during the stimulus

of increased pulmonary blood flow. These novel observations suggest that ET-1 may play an important

role in regulating lung growth in both normal and pathologic situations. Although age-matched control

lambs with lower pulmonary arterial pressures have similar alveolar growth as vehicle-treated shunt

lambs12, our results cannot rule out that the effect on alveolarization in the ET antagonist group was not

secondary to some indirect, hemodynamic effect. Therefore, further investigations will be required to

elucidate the role of ET-1 in regulating lung development. It is likely that such studies will yield important

biologic and clinically relevant information.

ETA block and increased pulmonary blood flow 13

GRANTS

This research was supported by grants HL61284 (JRF), HL48536 (BM), HL60190, and HL67841 (SMB)

from the National Institutes of Health and grant S/05/01 (SF) from the Deutsche Herzstiftung.

DISCLOSURES

There are no potential conflict of interests.

ETA block and increased pulmonary blood flow 14

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8. Vincent JA, Ross RD, Kassab J, Hsu JM, Pinsky WW. Relation of elevated plasma endothelin in congenital heart disease to increased pulmonary blood flow. Am J Cardiol. 1993;71:1204-7.

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13. Wong J, Reddy VM, Hendricks-Munoz K, Liddicoat JR, Gerrets R, Fineman JR. Endothelin-1 vasoactive responses in lambs with pulmonary hypertension and increased pulmonary blood flow. Am J Physiol. 1995;269:H1965-72.

14. Black SM, Bekker JM, Johengen MJ, Parry AJ, Soifer SJ, Fineman JR. Altered regulation of the ET-1 cascade in lambs with increased pulmonary blood flow and pulmonary hypertension. Pediatr Res. 2000;47:97-106.

15. Rabinovitch M, Haworth SG, Castaneda AR, Nadas AS, Reid LM. Lung biopsy in congenital heart disease: a morphometric approach to pulmonary vascular disease. Circulation. 1978;58:1107-22.

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16. Reid LM. The pulmonary circulation: remodeling in growth and disease. The 1978 J. Burns Amberson lecture. Am Rev Respir Dis. 1979;119:531-46.

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18. Black SM, Bekker JM, McMullan DM, Parry AJ, Ovadia B, Reinhartz O, Lakshminrushimha S, Mata-Greenwood E, Steinhorn RH, Fineman JR. Alterations in nitric oxide production in 8-week-old lambs with increased pulmonary blood flow. Pediatr Res. 2002;52:233-44.

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ETA block and increased pulmonary blood flow 16

FIGURE LEGENDS

Figure 1:

PD156707 treatment has little effect on percent medial thickness of pulmonary arteries up to >

10,000 um external diameter. Values are mean±SD, n=5 placebo-treated shunt-lambs and n=5 PD156707-

treated shunt-lambs.

Figure 2:

TOP: Light micrographs showing an increase in alveolar profiles in PD156707-treated shunt-lambs

(right) as compared to placebo-treated shunt-lambs (left). Verhoeff’s elastin stain/ Van Gieson.

BOTTOM: Bar graph showing that the number of alveolar profiles per unit area in increased in PD

156707-treated shunt lambs compared to placebo-treated shunt lambs. Values are Mean±SD, n=8 placebo-

treated shunt-lambs and 6 PD156707-treated shunt-lambs. *p = 0.002 vs. placebo.

ETA block and increased pulmonary blood flow 17

Figure 1:

0

1

2

3

4

5

6

PercentMedial

Thickness

External Diameter (µM)

0-50 -100 -150 -200 -300 -400 -500 -1000 >1000

PlaceboPD 156707

ETA block and increased pulmonary blood flow 18

Figure 2:

Top

Bottom

0

50

100

150

200

250

# AlveolarProfiles

Per Unit Area

Placebo

PD 156707 *

ETA block and increased pulmonary blood flow 19

TABLE 1: GENERAL HEMODYNAMIC VARIABLESHemodynamic Variable Placebo (n=8) PD156707 (n=6)

mPAP (mmHg) 25.0 ± 7.4 18.3 ± 8.5

mSAP (mmHg) 55.9 ± 8.5 54.5 ± 6.5

pulse pressure SAP (mmHg) 93.1 ± 21.8 55.0 ± 17.3 *

mPAP : mSAP 0.45 ± 0.1 0.33 ± 0.1

mRAP (mmHg) 5.4 ± 0.8 4.0 ± 1.2 *

mLAP (mmHg) 9.9 ± 2.9 7.3 ± 1.9

Qlpa (ml/min/kg) 0.13 ± 0.1 0.14 ± 0.1

PVRleft (mmHg/ml/min/kg) 118.1 ± 32.0 76.5 ± 34.1 *

SVR (mmHg/ml/min/kg) 416.3 ± 229.6 450.3 ± 205.0

Qp : Qs 2.43 ± 0.8 2.85 ± 0.6

mRAP: mean right atrial pressure, mPAP: mean pulmonary arterial pressure, mLAP: mean left atrial

pressure, mSAP: mean systemic arterial pressure, Qlpa: blood flow through the left pulmonary artery,

PVRleft: left pulmonary vascular resistance, SVR: systemic vascular resistance, Qp:Qs: ratio of pulmonary

to systemic blood flow. Values are mean±S.D., * p < 0.05 vs. placebo.

ETA block and increased pulmonary blood flow 20

TABLE 2: BIOCHEMICAL DETERMINATIONS

Plasma

NOx

(µM)

Tissue

NOx

(µM)

Plasma

cGMP

(pmol/ml)

Tissue

cGMP

(pmol/mg)

Plasma ET-1

(pg/ml)

NOS

Activity

(pmols/min/

mg protein)

PLACEBO 11.09±1.5 0.89±0.4 415.0±203 147.5±80.7 14.4±6.6 0.43±.0.09

PD156707 6.73±1.8* 0.59±0.5 672.8±418 148.1±63.5 23.3±9.5 0.45±0.04

Values are mean±SD, n=8 placebo-treated shunt-lambs and n=6 PD156707-treated shunt-lambs. * p <

0.05 vs. placebo.

ETA block and increased pulmonary blood flow 21

TABLE 3: PROTEIN DETERMINATIONS (DENSITOMETRIC VALUES)

eNOS PreproET-1 ECE-1

ETA Receptor ETB Receptor

PLACEBO 63.2±15.7 21.9±9.6 4130.6±1148.0 224585.7±478183 27401.6±14094

PD 156707 52.2±11.8 30.7±14.7 3988.3±1273.8 232175.7±375440 33196.6±19536

Values are mean±SD, n=8 placebo-treated shunt-lambs and n=6 PD156707-treated shunt-lambs. * p <

0.05 vs. placebo.

ETA block and increased pulmonary blood flow 22

TABLE 4: Percentage of Fully Muscular and Nonmuscular Pulmonary Arteries Related to Intraacinar Airway Level in PD156707 and Placebo-Treated Shunt-Lambs

Respiratory Bronchiole Alveolar Duct Alveolar Wall

Degree of Muscularization

% Distribution

Degree of Muscularization

% Distribution

Degree of Muscularization

% Distribution

muscular 98 ± 5.6 muscular 48 ± 16.9* muscular 27 ± 9,1*PD156707non-muscular 2 ± 5.6 non-muscular 52 ± 16.9* non-muscular 73 ± 9.1*

muscular 85 ± 14.6 muscular 22 ± 14.4 muscular 9 ± 7.4Placebonon-muscular 15 ± 14.6 non-muscular 78 ± 14.4 non-muscular 91 ± 7.4

Values are mean ± SD, n = 5 PD156707-treated shunt-lambs and n = 5 placebo-treated shunt-lambs. *p < 0.05 vs. placebo.