Endothelin-1 stimulation of endothelial nitric oxide synthase
in the pathogenesis of hepatopulmonary syndrome
Ming
Zhang1,
Bao
Luo1,
Shi-Juan
Chen2,
Gary A.
Abrams1, and
Michael B.
Fallon1
1 Department of Internal
Medicine, Liver Center, and
2 Vascular Biology and
Hypertension Program, University of Alabama at Birmingham, Birmingham,
Alabama 35294
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ABSTRACT |
Biliary cirrhosis
in the rat triggers intrapulmonary vasodilatation and gas exchange
abnormalities that characterize the hepatopulmonary syndrome. This
vasodilatation correlates with increased levels of pulmonary
microcirculatory endothelial nitric oxide synthase (eNOS) and hepatic
and plasma endothelin-1 (ET-1). Prehepatic portal hypertension induced
by portal vein ligation (PVL) does not cause similar changes,
suggesting that ET-1 in cirrhosis may modulate pulmonary eNOS and
vascular tone. We assessed whether ET-1 altered eNOS expression and
nitric oxide production in bovine pulmonary artery endothelial cells
(BPAECs) and if a 2-wk low-level intravenous ET-1 infusion in PVL
animals modulated pulmonary eNOS levels, microcirculatory tone, and gas
exchange. ET-1 caused a 2.5-fold increase in eNOS protein in BPAECs,
inhibitable with an endothelin B receptor antagonist, and an increase
in eNOS mRNA and nitrite production. ET-1 infusion in PVL animals
caused increased pulmonary eNOS levels, intrapulmonary vasodilatation,
and gas exchange abnormalities without increasing pulmonary arterial
pressure. ET-1 produced during hepatic injury may contribute to the
hepatopulmonary syndrome by modulating eNOS and inducing pulmonary
microcicrulatory vasodilatation.
endothelium; vasorelaxation; pulmonary; cirrhosis; expression
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INTRODUCTION |
DYSREGULATION OF the vascular endothelium plays a
fundamental role in a number of cardiovascular diseases (39) and in the altered vascular tone associated with cirrhosis and portal hypertension (37). An imbalance in the local production and/or activity of potent
vasoactive agents, including nitric oxide (NO) and endothelin-1 (ET-1),
in the vessel wall is thought to underlie endothelial dysfunction and
contribute to changes in vascular tone (8, 15, 28, 38). In models of
chronic liver disease, splanchnic vasodilatation is accompanied by
elevated endothelial nitric oxide synthase (eNOS) levels (28) and
enhanced NO activity (30), although the stimuli responsible for
increasing nitric oxide synthase (NOS) expression and NO production
remain controversial (37). Plasma ET-1 levels are also increased in
some forms of experimental and human cirrhosis and are postulated to
result from enhanced production in the damaged liver (3, 25, 26, 29,
31). ET-1 is a potent vasoconstrictor when acting through the
ETA receptor on the vascular
smooth muscle cells, although elevated levels in cirrhosis occur in the
setting of systemic vasodilatation and are not associated with
measurable vasoconstrictive effects (25). These observations suggest
that the effects mediated by ET-1 in chronic liver disease may include
stimulation of NOS activity through the
ETB receptor on endothelial cells
(17) or modulation of vasoactive peptide expression (4, 5).
The hepatopulmonary syndrome (HPS) is one well- recognized complication
of chronic liver disease that occurs in the setting of cirrhosis with
portal hypertension. It is characterized by intrapulmonary
vasodilatation, which results in altered arterial oxygenation
independent of intrinsic cardiopulmonary disease (23). In previous
studies, we have established chronic common bile duct ligation (CBDL)
in the rat as a model of HPS in which intrapulmonary vasodilatation
occurs in the setting of hepatic injury and portal hypertension (11).
In contrast, portal hypertension induced by partial portal vein
ligation (PVL) does not cause hepatic injury or HPS, suggesting that
portal hypertension alone may be a necessary but insufficient factor in
the development of HPS. In CBDL animals, the degree of intrapulmonary
vasodilatation and gas exchange abnormalities correlate with a
progressive increase in pulmonary vascular eNOS protein levels and
enhanced production and activity of NO in intralobar pulmonary rings
(10). Recently, we have also observed a progressive increase in hepatic
production and plasma levels of ET-1 after CBDL in the absence of
altered pulmonary ET-1 levels, which correlate with pulmonary eNOS
alterations and gas exchange abnormalities (26). These studies suggest
that enhanced ET-1 production during chronic hepatic injury may alter
pulmonary eNOS production and contribute to the onset of intrapulmonary
vasodilatation in experimental HPS.
In the present study, we test the hypothesis that low levels of
circulating ET-1, arising during chronic hepatic injury, induce pulmonary eNOS expression and increased NO production and contribute to
the development of intrapulmonary vasodilatation and HPS. Bovine pulmonary artery endothelial cells (BPAECs) are used to assess whether
ET-1 administration can trigger eNOS expression and enhanced NO
production in vitro and to assess the receptor specificity of the
effect. Chronic low-dose intravenous infusion of ET-1 is administered
to PVL animals to determine if ET-1 exposure, in the presence of portal
hypertension alone, results in increased pulmonary eNOS levels,
intrapulmonary vasodilatation, and arterial gas exchange abnormalities
consistent with the development of HPS.
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METHODS |
Animal models.
PVL was performed as previously described (7) using male Sprague-Dawley
rats (Charles River, Wilmington, MA) weighing 200-250 g. At the
time of surgery, a miniosmotic pump (ALZET Model 2002, ALZA, Palo Alto,
CA) was inserted into the femoral vein. Rats were infused with 3 ng · 200 g body
wt
1 · h
1
of synthetic ET-1 peptide (Peninsula Laboratories, Belmont, CA) or
0.9% saline for 2 wk based on the hepatic venous and plasma ET-1 concentration previously observed in 2-wk CBDL animals (26) and on
pilot studies. In pilot studies, the acute effects of ET-1 infusion
using 3 ng · 200 g body
wt
1 · h
1
was compared with an infusion of 30 ng · 200 g body
wt
1 · h
1.
The lower dose resulted in a mild transient hypotensive effect as
previously seen using physiological doses of ET-1, and the higher dose
resulted in well-described vasoconstriction (21). ET-1 delivery was
monitored by ensuring that the pump was empty and the delivery catheter
was positioned in the femoral vein at the termination of the
experiment. The stability of ET-1 in miniosmotic pumps over 2 wk was
documented by placing a known concentration of ET-1 in four miniosmotic
pumps under sterile conditions and incubating these pumps for 2 wk at
37°C. Subsequent RIA for ET-1 revealed a concentration of ET-1 in
the pumps within 10-15% of initial values. The development of
portal hypertension was confirmed by portal pressure and spleen weight
measurements. Arterial gas exchange was evaluated as described by
arterial blood gas analysis (11), and the alveolar-arterial oxygen
gradient was calculated as
150
(PCO2/0.8)
PO2.
Lung tissue was collected after perfusion with PBS for histology and
eNOS detection by Western blot analysis (10). Mean pulmonary arterial
pressure was measured by insertion of an indwelling pulmonary arterial
catheter, and mean systemic arterial pressure was measured by insertion
of an indwelling femoral arterial catheter both placed 48 h before
death as previously described (9). Pressure measurements were made at
rest on the day of death. Five to six animals were used in each group.
The study was approved by the Institutional Animal Care and Use
Committee of the University of Alabama at Birmingham and conforms to
the National Institutes of Health Guidelines for the
Care and Use of Laboratory Animals.
Microsphere protocol.
Size assessment of the pulmonary microcirculation was performed as
previously described in awake unsedated animals (11). Forty-eight hours
before microsphere injection, animals underwent the placement of
indwelling PE-50 femoral arterial and venous catheters. On the day of
measurement, 45 min after arterial blood gas determination, 2.5 × 10 custom mixed and counted cross-linked polystyrene-divinylbenzene
microspheres labeled red (size range 6.5-10 µm; Interactive
Medical Technologies, Los Angeles, CA) in 0.20 ml of sterile PBS were
injected over 2-4 s through the femoral vein catheter, which was
immediately flushed with 0.2 ml of sterile PBS over 2-4 s. An
aliquot of microspheres was removed from the injection syringe
immediately before injection and counted to verify the numbers and
sizes of microspheres injected. A reference blood sample was withdrawn
from the femoral arterial catheter beginning at the time of femoral
vein injection for a total of 90 s at a constant rate of 1.0 ml/min.
The volume removed was replaced with an equal volume of sterile PBS.
Microsphere counting/intrapulmonary shunt calculations.
Samples of beads before venous injection and reference blood samples
were coded, and counting suspensions were prepared using the E-Z Trac
method as outlined by the manufacturer. Numbers and sizes of
microspheres in each sample were assessed using a Leica DMRE microscope (Wetzlar, Germany) with a color video
imaging system combined with image analysis software (Pro 3.0 Media
Cybernetics, Silver Spring, MD) with area, shape factor, and aspect
ratios set to distinguish microsphere sizes. Total numbers of
microspheres passing through the pulmonary microcirculation were
calculated as reference blood sample microspheres per milliliter times
estimated blood volume. Blood volume of each animal was derived from
the following formula: blood volume (ml) = 0.06 × body wt (g) + 0.77 (see Ref. 24). Intrapulmonary shunting was calculated as an intrapulmonary shunt fraction (%) = (total number of microspheres passing through the pulmonary microcirculation/total beads injected into the venous circulation) × 100.
Cell culture.
BPAECs [American Type Culture Collection (ATCC), Manassas,
VA] were maintained and passaged in EGM-MV complete
media (Clonetics, San Diego, CA) at 37°C with 5%
CO2 on 100-mm Falcon cell culture dishes (Becton Dickinson Labware, Lincoln Park, NJ), and cells of
passages 3-10 were used in the
study. When cells reached 70% confluence, they were incubated in 4 ml
of minimal media containing 0.5% FBS without supplements and
stimulated by ET-1 at various concentrations (0.01, 0.1, and 1 µM)
for various times (12, 24, and 48 h). In a set of experiments,
endothelin receptor antagonists including TBC3214Na for
ETA (kind gift from Dr. Y. F. Chen, University of Alabama at Birmingham), BQ-788 for
ETB (Peptides International, Louisville, KY), or Bosentan for
ETA and
ETB (Roche, Basel, Switzerland) were applied at 10 µM to cells in the presence or absence of ET-1 treatment. To assess cell proliferation, cells were incubated on a
96-well plate (Nalge Nunc, Naperville, IL) at 2,000 cells/well in
minimal media containing 0.5% FBS in the presence or absence of 0.1 µM ET-1 for 12, 24, and 48 h. A standard curve was constructed by
using a defined cell number in the assay. At each time point, 20 µl
of freshly made MTS/phenazine methosulfate (PMS) mixture (10:1 vol/vol,
Promega, Madison, WI) was added into each well containing 100 µl of
media and was incubated at 37°C in a humidified 5%
CO2 atmosphere for 1 h. After the
absorbance at 490 nm was recorded using an ELISA microplate reader
(Molecular Devices, Sunnyvale, CA), cell number was calculated
according to the standard curve.
Western blot analysis.
BPAECs or lung tissue was prepared by lysis or Dounce homogenization
respectively in radioimmunoprecipitation buffer (10) in the presence of
protease inhibitors. Fifteen micrograms of protein from homogenates,
quantified by protein assay (Bio-Rad, Hercules, CA), were fractionated
on a 7.5% SDS-PAGE gel and then transferred to a Hybond enhanced
chemiluminescence nitrocellulose membrane (Amersham, Arlington Heights,
IL). Incubation with an eNOS monoclonal antibody (Transduction
Laboratories, Lexington, KY) was followed by extensive washing and
incubation with a horseradish peroxidase-conjugated sheep anti-mouse
secondary antibody (Amersham) and development by enhanced
chemiluminescence (Amersham) on Kodak X-ray film (Sigma, St. Louis, MO).
Northern blot analysis.
Total RNA from BPAECs was prepared by lysis in TRIzol reagent on the
basis of a standard protocol (GIBCO, Grand Island, NY). RNA (20 µg) was subjected to agarose gel electrophoresis and blotted onto a Nytran membrane (Schleicher & Schuell, Keene, NH) as described (26), followed by a quick hybridization procedure (Stratagene, La
Jolla, CA) with a 4.0-kb bovine eNOS cDNA probe (kind gift of Dr.
William Sessa, Yale University) or a 0.5-kb rat
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (ATCC) labeled
with the Prime-a-Gene labeling system (Promega). Blots were then
exposed to Kodak X-ray film.
RT-PCR.
First strand cDNA was synthesized from 2 µg of total RNA of BPAECs by
oligo(dT) primer using the SuperScript preamplification system (GIBCO)
and then subjected to subsequent PCR analysis. PCR was carried out
using appropriate primers in 50 µl of buffer containing 0.2 mM each
dNTP, 3.0 mM MgCl2, 0.5 µM each
primer, and 2.5 U/100 µl recombinant
Taq DNA polymerase (GIBCO) using 25 amplification cycles of 94°C for 30 s, 55°C for 30 s, and
72°C for 1 min in a Perkin-Elmer 2400 PCR machine (Foster City,
CA). The reaction mixture was then resolved by agarose gel
electrophoresis. PCR primers for detecting eNOS mRNA were designed
based on the published eNOS cDNA sequence to yield a ~380-bp DNA
fragment (20) and were also used in subsequent sequence analysis. The
5' primer was AGTAACACAGACAGTGCAGGG, and the 3' primer was
GTAGCCTGGAACATCTTCCG. Primers for detecting
-actin message were used
as an internal control to amplify a ~400-bp DNA fragment. The
5' primer was GACCTGACAGACTACCTCAT, and the 3' primer was
AGACAGCACTGTGTTGGCAT. Where indicated, PCR products were sequenced
using an ABI PRISM Model 377 automated sequencer (University of Alabama
at Birmingham Sequencing Facility).
NO detection assay.
Aliquots of media (150 µl) from BPAEC culture in the presence or
absence of 0.1 µM ET-1 for 0, 12, and 24 h were incubated with 10 µl of Escherichia coli nitrate
reductase preparation (no. 25922, ATCC) at 37°C for 1 h to convert
NO
3 in samples to
NO
2, followed by centrifugation at
15,000 g for 5 min. Supernatants (150 µl) were then analyzed by the Greiss reaction (14) in 75 µl of 1%
sulfanilamide and 7 µl of naphthylethylene-diamine
dihydrochloride at room temperature for 10 min. The absorbance at 550 nm was measured using an ELISA microplate reader, and net NO production
was calculated according to a standard curve prepared by using
NaNO3 ranging from 0 to 100 µM
in culture media.
Densitomertry and statistics.
The density of autoradiographic signals was quantitated with a GS-670
imaging densitometer (Bio-Rad) and expressed as multiples of control in
which the density was designated as onefold in control experimental
group. Data were analyzed using Student's
t-test or ANOVA with Bonferroni
correction for multiple comparisons between groups. All measurements
are expressed as means ± SE. Statistical significance was
designated as P < 0.05 unless
otherwise indicated.
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RESULTS |
ET-1 increases eNOS protein levels in BPAECs.
To determine if ET-1 can induce eNOS protein expression, BPAECs were
treated with synthetic ET-1 peptide in a minimal medium containing
0.5% FBS in the absence of other growth factor supplements. A
progressive dose- and time-dependent increase in eNOS protein (140 kDa)
levels was observed by Western blotting after ET-1 stimulation. The
maximal increase occurred at 24 h after the addition of 0.1 µM ET-1 (Fig. 1,
A and
B) and represented a significant
2.5-fold increase in eNOS protein production (Fig.
1C).

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Fig. 1.
Endothelin (ET)-1 induction of endothelial nitric oxide synthase (eNOS)
protein levels in bovine pulmonary artery endothlial cells (BPAECs).
BPAECs (70% confluent) were treated with ET-1 peptide of varying doses
and for various times and then lysed in radioimmunoprecipitation (RIPA)
buffer. Equal amounts of lysates were resolved on 7.5% SDS-PAGE gels
and transferred to Hybond ECL membranes. eNOS was detected with a
monoclonal antibody and visualized by enhanced chemiluminescence (ECL).
A: BPAECs stimulated with 0.1 µM
ET-1 for 0, 12, 24, or 48 h demonstrated an increase in eNOS protein
levels. B: BPAECs stimulated with ET-1
at 0, 0.01, 0.1, or 1 µM for 24 h demonstrated a dose-dependent
increase in eNOS protein levels. C:
summary of BPAEC stimulation for 24 h in presence and absence of 0.1 µM ET-1 (n = 5 plates for each
group). eNOS protein was quantitated by desitometry, and values are
expressed as means ± SE. Mean for non-ET-1-treated cells was
arbitrarily set at 1. * P < 0.05.
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ET-1-mediated effects on eNOS protein are mediated through the
ETB receptor in BPAECs.
The effects of ET-1 on vascular tone are mediated through two distinct
receptors, the ETA receptor and
the ETB receptor, which differ in
their tissue and cell-type specificity (19). The
ETB receptor predominates on
endothelial cells. To determine the role of ET receptors in
ET-1-mediated alterations in eNOS protein levels, BPAECs were
stimulated with 0.1 µM ET-1 for 24 h and eNOS protein levels were
assessed in the presence or absence of ET receptor antagonists (Fig.
2). The eNOS protein levels in these cells
were compared with control levels in untreated BPAECs, which were
arbitrarily set at one. The addition of ET receptor antagonists alone,
including TBC3214Na, a selective
ETA receptor antagonist (1.0 ± 0.2-fold control), BQ-788, a selective
ETB receptor antagonist (1.2 ± 0.06-fold control), and Bosentan, a mixed
ETA and
ETB receptor antagonist (1.1 ± 0.2-fold control), had no significant effect on eNOS protein levels
(n = 4 in each group and in subsequent
receptor antagonist studies; differences were not significant for each
group relative to untreated controls). The addition of the
ETA receptor antagonist did not
inhibit the ET-1-mediated increase in eNOS protein (2.3 ± 0.2-fold
control; P < 0.05 relative to
control untreated cells), which was similar to that seen in control
BPAECs treated with ET-1 alone. In contrast, the ET-1-mediated
increase in eNOS protein was abolished and was significantly
different from ETA receptor antagonist-treated cells when the
ETB receptor antagonist (1.03 ± 0.3-fold control) or Bosentan (1.03 ± 0.04-fold control) was added to the culture medium (P < 0.05 relative to ETA receptor antagonist-treated cells). These findings indicate that ET-1-mediated changes in eNOS protein levels are mediated through the
ETB receptor on BPAECs.

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Fig. 2.
Effects of endothelin receptor antagonists on ET-1-induced eNOS protein
levels in BPAECs. Representative Western blot for eNOS obtained from
70% confluent BPAECs treated with 10 µM of endothelin receptor
antagonists for 24 h in presence or absence of 0.1 µM ET-1 and
analyzed as described in Fig. 1. TBC3214Na is
ETA receptor antagonist, BQ-788 is
ETB receptor antagonist and
Bosentan is ETA and
ETB receptor antagonist.
Experiments were repeated (n = 4 for
each condition) for statistical analysis. Second, fourth, and sixth
lanes from left represent cells incubated with ET receptor antagonists
alone. No difference in eNOS levels was observed in these cells
compared with untreated control cells (first lane from left).
Incubation of cells with a selective
ETA receptor antagonist and ET-1
did not block ET-1-mediated increase in eNOS protein levels (third lane
from left). In contrast, both
ETB-selective and mixed
ETA and
ETB receptor antagonists abolished
ET-1-mediated increase in eNOS protein levels (fifth and seventh lanes
from left), establishing that ET-1 alters eNOS protein levels in BPAECs
through ETB
receptor.
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ET-1 upregulates eNOS mRNA in BPAECs.
To further evaluate the mechanism of the ET-1-mediated increase in eNOS
protein, we measured steady-state eNOS mRNA levels by Northern blotting
in BPAECs after ET-1 exposure (Fig.
3A). The
eNOS mRNA levels normalized to GAPDH levels after ET-1 exposure were
compared with control levels in untreated BPAECs incubated for the same
time as treated cells and arbitrarily set at 1 (n = 4 for each group). There was an
increase in the ~5.0-kb eNOS signal (2) that was statistically
significant within 12 h of 0.1 µM ET-1 stimulation (2.3 ± 0.6-fold control) and was maximal by 24 h (3.6 ± 1.2-fold control).
The increase was maintained at 48 h (2.9 ± 0.6-fold control). No
change in the GAPDH control signal was observed on these blots. The
ET-1-mediated increase in eNOS mRNA was confirmed by RT-PCR analysis
using eNOS-specific primers. An increase in a ~380-bp fragment
amplified with eNOS primers, but not an
-actin control message, was
observed after ET-1 stimulation (Fig.
3B). The ~380-bp PCR product was
confirmed to be eNOS by direct sequencing and comparison with the
published eNOS sequence (data not shown) (20). The temporal association between the increase in eNOS mRNA and protein levels after ET-1 exposure is consistent with the hypothesis that ET-1 alters eNOS expression in endothelial cells.

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Fig. 3.
ET-1 induction of eNOS mRNA in BPAECs. BPAECs (70% confluent) were
stimulated with 0.1 µM ET-1 peptide for 0, 12, 24, or 48 h, and total
RNA was extracted using TRIzol reagent.
A: representative Northern blot of 20 µg of total RNA fractionated on a 0.75% formaldehyde-agarose gel,
transferred to nitrocellulose, and detected with a 4.0-kb eNOS probe. A
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe was used as
an internal control. The ~5.0-kb eNOS mRNA increased maximally by 24 h with no change in GAPDH signal. B: 2 µg of total RNA from BPAECs stimulated by 0.1 µM ET-1 for 0, 12, 24, or 48 h were subjected to RT-PCR using specific primers for eNOS or
-actin and then resolved on a 0.75% agarose gel. The 380-bp eNOS
PCR product also increased after ET-1 stimulation, with no change in
-actin PCR product.
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ET-1 stimulates NO production in BPAECs.
To confirm that ET-1 stimulation of BPAECs and the subsequent increase
in eNOS protein levels result in enhanced NO production, we measured NO
levels in BPAEC culture media in the presence and absence of exogenous
ET-1. ET-1 administration (0.1 µM) significantly stimulated net NO
production (2.8-fold) from cells after 24 h (Fig.
4), as has been previously observed (17).
The maximal increase in culture medium NO levels correlated temporally
with increased eNOS levels in BPAECs, suggesting that enhanced eNOS expression may contribute to increased NO production.

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Fig. 4.
ET-1 induction of nitrite production in BPAECs. After incubation in
presence or absence of 0.1 µM ET-1 for 0, 12, or 24 h,
NO 3 in BPAEC culture media was
converted to NO 2 by nitrate reductase
and then analyzed by Greiss reaction. Absorbance at 550 nm was measured
to calculate net NO production according to a standard curve. A
significant increase in nitrite was observed at 24 h after ET-1
induction (n = 4 at each time point in
each group). * P < 0.05.
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ET-1 does not induce BPAEC proliferation over 24 h.
To exclude the possibility that the mitogenic properties of ET-1 caused
BPAEC proliferation and resulted in increased eNOS mRNA and protein
levels over the time course studied here, we measured BPAEC
proliferation by the MTS/PMS assay in the presence and absence of
exogenous ET-1. Cells incubated as above and not exposed to ET-1 were
compared with cells exposed to 0.1 µM ET-1 for 12, 24, and 48 h. Cell
number was measured at each time point. There was no significant
difference in cell number after 24 h between exposed and unexposed
cells (Fig. 5). After 48 h, ET-1 treatment
resulted in a modest increase in cell number, though the magnitude of
the increase relative to untreated cells at 48 h (1.2-fold untreated)
was substantially less than the increase in eNOS mRNA and protein
levels over the same time frame. These results demonstrate that the
increase in eNOS levels in BPAECs after ET-1 stimulation cannot be
explained by enhancement of cell proliferation over the time frame
studied here.

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Fig. 5.
Effect of ET-1 on proliferation of BPAECs. Two thousand cells/well in a
96-well plate were incubated at 37°C and 5%
CO2 atmosphere in a growth
factor-deficient minimal medium for 0, 12, 24, or 48 h in presence or
absence of 0.1 µM ET-1. Cell numbers were measured by MTS/phenazine
methosulfate assay and were calculated according to a standard curve
for each time point (n = 4 at each
time point).
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Chronic intravenous ET-1 administration increases lung eNOS levels,
results in intrapulmonary shunting, and impairs gas exchange after PVL.
To determine if ET-1 contributes to the development of HPS, we
delivered an ET-1 or saline infusion through the femoral vein over 2 wk
via a miniosmotic pump in PVL animals. Liver and lung histology, portal
vein pressure, mean systemic and pulmonary arterial blood pressure,
lung eNOS levels, microsphere assessment of the pulmonary
microcirculation, and arterial gas exchange were evaluated. Pressure
measurements and arterial blood gas results are summarized in Table
1. All animals developed significant portal
hypertension compared with values previously observed in normal rats
(11), and the degree of portal hypertension at 2 wk was not different in saline- and ET-1-treated animals. Serum aspartate aminotransferase and total bilirubin levels and hepatic histology were also normal in
the saline and ET-1 groups (data not shown), suggesting that low-dose
ET-1 infusion had no measurable effects on the liver. Mean systemic
arterial pressures were similar in saline- and ET-1-treated animals and
were lower than values we have observed in normal animals (9),
reflecting the development of a hyperdynamic state as previously
described after PVL (36). Mean pulmonary arterial pressures were also
similar in both groups and remained within the range observed in normal
animals (9), demonstrating that the doses of exogenous ET-1
administered here had no significant vasoconstrictive effect in the
lung.
Western blot analysis of pulmonary eNOS protein levels demonstrated a
small but significant 1.5-fold increase in whole lung eNOS levels in
ET-1-treated lung relative to saline-treated lung (Fig.
6). This increase is similar but slightly
less than that observed in 2-wk CBDL animals with HPS as previously
described (10). Western blot for inducible nitric oxide synthase was
performed and revealed no detectable signal in lung in either group
(data not shown) as observed previously in CBDL animals (10). The increase in pulmonary eNOS levels in ET-1-treated animals was accompanied by increased shunting of microspheres across the pulmonary microcirculation and significant alterations in arterial gas exchange. Figure 7A
demonstrates that similar numbers and sizes of microspheres were
injected into the venous system in saline- and ET-1-treated animals.
Figure 7B shows the results of
arterial counts of microspheres after venous injection, reflecting
passage through the pulmonary microcirculation. Saline-treated PVL
animals demonstrated a similar pattern of microsphere passage through
the lungs as previously observed in normal and PVL animals (11).
ET-1-treated animals demonstrated a significant increase in the size
and number of microspheres passing through the lungs relative to
saline-treated animals, as evidenced by a rightward shift in the
microspheres recovered from the femoral artery in these animals.
Saline-treated PVL animals had a shunt fraction of 2.6 ± 0.8%,
similar to shunt values found for 6.5 × 10 µm microspheres in
normal and PVL animals in previous studies (11). In contrast,
ET-1-treated animals had a significantly increased shunt fraction of
14.7 ± 2% (P < 0.05) similar to
that previously observed in CBDL animals with HPS (11), reflecting
intrapulmonary vasodilatation and enhanced passage of microspheres
through the lung.

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Fig. 6.
Effect of chronic ET-1 infusion on lung eNOS protein levels in PVL
rats. Lung tissue from PVL rats receiving intravenous saline or
low-dose ET-1 (3 ng · 200 g body
wt 1 · h 1)
through miniosmotic pumps for 2 wk were homogenized in RIPA buffer, and
15 µg of total lysates were subjected to immunoblotting as described
in Fig. 1. Specific eNOS protein signal was quantified by densitometry
and expressed as mean ± SE. Mean from saline-treated rats was
arbitrarily set at 1, and ET-1-treated values were expressed as fold
control (n = 4 in each group). A
significant increase in lung eNOS protein levels occurred after ET-1
infusion. * P < 0.05.
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Fig. 7.
Effect of chronic ET-1 infusion on pulmonary micro-circulation as
measured using microsphere analysis. PVL rats that had received chronic
saline or ET-1 intravenous infusions through miniosmotic pumps for 2 wk
were injected with 2.5 × 106
custom mixed and counted cross-linked polystyrene-divinylbenzene
microspheres labeled red (size range 6.5-10 µm) via an
indwelling femoral vein catheter, followed by collection of blood from
an indwelling femoral arterial catheter. Numbers and sizes of
microspheres were assessed using a microscope equipped with a color
video imaging system and image analysis software.
A: numbers and sizes of microspheres
injected into femoral vein measured by counting an aliquot of
microspheres removed from syringe immediately before injection. Similar
numbers and sizes of microspheres were injected in both groups.
B: numbers and sizes of microspheres
collected from femoral artery over 90 s after venous injection. A
significant increase in numbers and sizes of microspheres were
recovered in femoral artery in ET-1-infused animals compared with
saline-infused animals, reflecting increased passage through dilated
pulmonary microcirculation. * P < 0.05 (n = 4).
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Evidence of dilatation of the pulmonary microcirculation was also
accompanied by abnormalities in arterial oxygenation in ET-1-treated
animals (Table 1). Compared with animals without liver
disease and to untreated PVL animals (11), each ET-1-treated PVL animal
developed a respiratory alkalosis, relative hypoxemia, and a widened
alveolar-arterial oxygen gradient, similar to changes observed in
animals and humans with HPS (10). An arterial blood gas was available
from one saline-treated PVL animal, and values from this animal were
within the normal range that we have previously observed (11). These
observations demonstrate that chronic low-dose ET-1 infusion in PVL
animals results in the development of molecular alterations in lung
eNOS, dilatation of the pulmonary microcirculation, and gas exchange
abnormalities similar to changes observed in animals with HPS.
 |
DISCUSSION |
Reciprocal paracrine and autocrine interactions between ET-1 and NO are
important in mediating endothelium-dependent regulation of vascular
tone (8, 15, 38, 39). An imbalance in these mediators has been
implicated in the pathogenesis of several vascular disorders (13, 18,
39). The present study extends this concept on the basis of our
previous observations, which suggest a pathogenetic association between
elevated circulating ET-1 levels, increased pulmonary vascular eNOS
levels, and functional alterations in the pulmonary vasculature in an
animal model of HPS (26). In the present study, we document that
administration of ET-1 stimulates increased eNOS protein and mRNA
levels in BPAECs through an ETB receptor-mediated effect, which correlates with increased NO production in these cells. In addition, chronic low-dose intravenous infusion of
ET-1 in PVL animals triggers an increase in lung eNOS levels, alterations in the pulmonary microcirculation, and an impairment in
arterial gas exchange similar to that observed in animals with HPS,
without increasing pulmonary arterial pressure. These studies provide
direct evidence that circulating ET-1 may increase eNOS production in
endothelial cells and contribute to the pathogenesis of HPS.
We and others have demonstrated that hepatic production of ET-1
increases after CBDL (26, 34) and likely results in release into the
venous system with circulation to the pulmonary vasculature. The
increase in circulating ET-1 correlates with increased eNOS levels,
enhanced NO activity, and gas exchange abnormalities in the lung in
these animals that develop HPS (26) and implies that ET-1 may
contribute to the development of pulmonary abnormalities. Our in vitro
studies demonstrate the novel observation that exposure to ET-1 does
increase eNOS mRNA and protein levels in BPAECs in a dose- and
time-dependent fashion, supporting a role for ET-1 in altering eNOS
expression. This effect occurs through the
ETB receptor. Although the
molecular mechanism through which ET-1 alters eNOS production remains
under study, the hypothesis that ET-1 alters eNOS expression in the
pulmonary vasculature is consistent with the observation that ET-1 can
alter vasoactive mediator expression in other cell types (4, 5) and
with the finding that a variety of agents are now known to influence
the expression of the eNOS gene (6, 16, 22, 32). The potential
physiological significance of this effect is highlighted by our in vivo
observation that chronic low-dose intravenous ET-1 infusion also
increases pulmonary eNOS levels and is associated with vascular and gas
exchange abnormalities in the lung.
ET-1 has potent vasoconstrictive properties when released from vascular
endothelial cells and targeted to the
ETA receptor on vascular smooth
muscle cells or when administered intravenously in pharmacological
doses (35). However, this effect is not observed when doses closer to
levels found in vivo are administered (21). The ET-1 peptide also
exerts an autocrine vasodilatory effect by increasing eNOS activity
through the ETB receptor on
vascular endothelial cells (17) and alters the gene expression of other vasoactive peptides in smooth muscle cells (4, 5). Liver injury with
portal hypertension appears to represent a unique pathological
situation where elevated circulating ET-1 levels (3, 26, 29), derived
from increased hepatic production (26, 31, 34), occur in the setting of
systemic vasodilatation. In this situation, systemic vascular tone is
not altered by the addition of ET receptor antagonists (25), suggesting
that ET-1 production does not simply reflect a response to
vasodilatation. Our in vivo findings demonstrate that low-dose
intravenous ET-1 infusion in the setting of portal hypertension does
not significantly alter systemic or pulmonary arterial pressure,
confirming that vasoconstrictive effects of ET-1 are not detectable.
The attenuated vasoconstrictive effect of circulating ET-1 in portal
hypertension is unexplained but may be contributed to by the relatively
low circulating levels observed, which are generally below levels associated with significant vasoconstriction (27). Alternatively, flow-mediated changes in ET receptor expression in the vasculature (33)
or alterations in the levels of other vasoactive substances observed
during the development of the hyperdynamic circulation of portal
hypertension could influence the effects of ET-1 on the vasculature.
Our studies also do not support that circulating ET-1 contributes to
the development or maintenance of systemic vasodilatation in portal
hypertension, as saline- and ET-1-treated PVL animals develop a similar
degree of systemic hypotension. Thus ET-1 appears to exert a selective
vasodilatory effect in the pulmonary vasculature in our system.
The observation that low levels of circulating ET-1 may have a
localized effect in the pulmonary vasculature that alters eNOS levels
and influences the pulmonary microcirculation is perhaps not
unexpected. ET-1 released into the circulation during hepatic injury or
infused intravenously will first encounter the pulmonary vasculature.
In addition, significant clearance of ET-1 from the circulation occurs
in the lung, in large part through an
ETB receptor-mediated effect (12).
Thus ET-1 may influence pulmonary vascular NO production by increasing
eNOS levels and by increasing eNOS enzyme activity, effects that are
each mediated through the ETB
receptor. Our in vitro and in vivo studies support this concept.
However, we cannot be certain that eNOS is not also produced in cell
types other than endothelial cells in the vascular wall or may increase
in cell types outside the vasculature in the lung. In addition, we have
not completely excluded that other forms of nitric oxide synthase may
be contributing to changes in pulmonary vascular tone, although we have
not found detectable levels of iNOS in the lung in the present studies.
Finally, the increase in lung eNOS levels observed after ET-1 infusion
is less than that observed in CBDL animals despite the development of a
similar degree of vasodilatation and gas exchange abnormalities. This
observation suggests that ET-1 may also contribute to intrapulmonary
vasodilatation through effects distinct from nitric oxide production in
the pulmonary vasculature.
In humans and animals the presence of portal hypertension appears to be
required for the development of HPS. Thus we designed our in vivo
studies to administer low-dose intravenous ET-1 to animals that
normally develop portal hypertension in the absence of pulmonary
vascular or hepatic changes to provide a direct means of analyzing
whether ET-1 triggers changes in the pulmonary microcirculation in the
setting of portal hypertension. We have evaluated the effects of
chronic intravenous ET-1 infusion on the liver as well as the lung in
PVL animals and have observed no effect on portal venous pressure,
liver tests, or hepatic histology. These results confirm a lack of
measurable effects of exogenous low-level ET-1 infusion in the liver in
PVL animals. Our findings in the lung are consistent with the concept
that chronic ET-1 infusion, in the setting of portal hypertension,
causes intrapulmonary vasodilatation. Studies evaluating ET-1 infusion
in normal animals will define whether portal hypertension is required
for ET-1 to alter pulmonary microvascular tone.
Our microsphere assessment of the pulmonary microcirculation provides a
direct assessment of small vessel tone and intrapulmonary shunting in
unsedated animals. The technique is an adaptation of methods used to
evaluate intrapulmonary shunting in humans with HPS (1) and
portosystemic shunting in anesthetized rats (7) and has been previously
employed in PVL animals (11). Results in our saline-treated PVL animals
in the present study are similar to those observed in PVL animals
previously (11), confirming the reproducibility of this technique. Our
ET-1-treated animals shunted significantly more and larger microspheres
across pulmonary microcirculation, similar to changes previously
observed in CBDL animals with HPS (11). One limitation of this
technique is the inability to discriminate between intrapulmonary and
intracardiac shunting of microspheres. Thus if ET-1 infusion resulted
in a significant selective increase in pulmonary arterial pressures, a
gradient for right-to-left intracardiac shunting of microspheres through potential intracardiac septal defects could be created. We did
not observe a significant rise in pulmonary arterial pressures in
ET-1-treated PVL animals and could find no gross anatomical evidence
for the presence of intracardiac septal defects in previous studies
(11) or in our present animals.
Our current findings, in conjunction with previous observations,
suggest that increased ET-1 production found during certain forms of
hepatic injury may stimulate pulmonary vascular eNOS expression and
activity and contribute to the pathogenesis of HPS. In vitro studies
demonstrate that this effect may be mediated through the
ETB receptor on pulmonary vascular
endothelial cells. Together, these observations provide a novel
pathogenetic mechanism for the development of HPS. In addition, they
provide a direction for future investigation in humans and imply that
unique therapeutic approaches focused on inhibiting the pulmonary
vascular ETB receptor or on
modulating hepatic ET-1 production may provide useful medical therapies
for this disorder.
 |
ACKNOWLEDGEMENTS |
This work was supported by a Merit Award from the Department of
Veterans Affairs (M. B. Fallon) and a Grant In Aid from the Alabama
affiliate of the American Heart Association (M. B. Fallon).
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. B. Fallon,
Univ. of Alabama at Birmingham, 410 LHRB, 701 South 19th St.,
Birmingham, AL 35294-0007 (E-mail:
mfallon{at}uab.edu).
Received 25 February 1999; accepted in final form 26 July 1999.
 |
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