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887
Contribution of Endogenous Endothelin-1 to the
Progression of Cardiopulmonary Alterations in
Rats With Monocrotaline-Induced
Pulmonary Hypertension
Takashi Miyauchi, Ryosuke Yorikane, Satoshi Sakai, Takeshi Sakurai, Megumu Okada,
Masaru Nishikibe, Mitsuo Yano, Iwao Yamaguchi, Yasuro Sugishita, Katsutoshi Goto
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Endothelin-1 (ET-1) is known to have potent contractile and proliferative effects on vascular smooth
muscle cells and is known to induce myocardial cell hypertrophy. We studied the pathophysiological role
of endogenous ET-1 in rats with monocrotaline-induced pulmonary hypertension. Four-week-old rats were
given a single subcutaneous injection of 60 mg/kg monocrotaline (MCT rats) or saline (control rats) and
were killed after 6, 10, 14, 18, and 25 days. In the MCT rats, right ventricular systolic pressure
progressively increased and right ventricular hypertrophy developed in a parallel fashion. The venous
plasma ET-1 concentration also progressively increased, and this increase preceded the development of
pulmonary hypertension. The isolated pulmonary artery exhibited a significantly weaker response to ET-1
in the MCT rats on day 25 but not on days 6 and 14. In the MCT rats, the expression of prepro ET-1
mRNA as measured by Northern blot analysis significantly increased in the heart on days 18 and 25,
whereas it gradually decreased in the lungs. The peptide level of ET-1 in the lungs also significantly
decreased in the pulmonary hypertensive stage. The expression of prepro ET-1 mRNA had increased by
day 6 only in the kidneys. Continuous infusion of BQ-123, a selective ETA receptor antagonist, by an
osmotic minipump (14.3 mg per day per rat for 18 days) significantly inhibited the progression of both
pulmonary hypertension (right ventricular systolic pressure, 77.8±4.2 [mean+ SEMI mm Hg [n=10]
versus 52.3+±2.4 mm Hg [n=7]; P<.01) and right ventricular hypertrophy (right ventricle/[left
ventricle±+septum], 0.56±0.03 [n=10] versus 0.41+0.02 [n=71; P<.01). Histological examination revealed that BQ-123 also effectively prevented pulmonary arterial medial thickening. The inhibition of
right ventricular hypertrophy by BQ-123 may be partly ascribed to the blockade of excessive stimulation
of the heart by ET-1, in addition to the prevention of pulmonary hypertension. The present findings
suggest that endogenous ET-1 contributes to the progression of cardiopulmonary alterations in rats with
MCT-induced pulmonary hypertension. (Circ Res. 1993;73:887-897.)
KEY WoRDs * endothelin-1 * pulmonary hypertensive rats * gene expression * ETA receptor
antagonist * plasma concentration
V arious cardiac diseases (eg, congenital heart
disease, valvular heart disease, and left ventricular failure) and pulmonary diseases (eg,
emphysema, lung fibrosis, and obstructive airway disease) are often accompanied by pulmonary hypertension, and the severity of pulmonary hypertension is one
determinant of the prognosis of such patients.' Although some researchers believe that pulmonary vasoconstriction plays a role in the pathogenesis of pulmonary hypertension,"2 the mechanism for the progression
of pulmonary hypertension is still poorly understood.
Received October 26, 1992; accepted July 13, 1993.
From the Department of Pharmacology (T.M., R.Y., S.S., T.S.,
K.G.), Institute of Basic Medical Sciences, and the Department of
Internal Medicine (I.Y., Y.S.), Institute of Clinical Medicine,
University of Tsukuba, Ibaraki, Japan; and Tsukuba Research
Institute (M.O., M.N., M.Y.), Banyu Pharmaceutical Co, Ltd,
Ibaraki, Japan.
Correspondence to Katsutoshi Goto, PhD, Department of Pharmacology, Institute of Basic Medical Sciences, University of
Tsukuba, Tsukuba, Ibaraki 305, Japan.
Endothelin-1 (ET-1), a potent endothelium-derived
vasoconstrictor peptide, was recently identified.3 This
peptide induces proliferation of vascular smooth muscle
cells.4 ET-1 has several properties suggestive of a potential pathophysiological role in pulmonary hypertension. First, ET-1 contracts isolated pulmonary vessels5
and increases pulmonary vascular resistance.6'7 Second,
ET-1 has a mitogenic effect on vascular smooth muscle
cells4'8'9 and fibroblasts,'0"' consistent with a role in
vascular remodeling, a prominent finding in pulmonary
hypertensive stages. ET-1 has also been reported to be
produced by nonvascular tissues such as the heart,
kidneys, and central nervous system.'2"3 It has been
demonstrated that prepro ET-1 mRNA is expressed in
cultured rat cardiomyocytes'4 and that ET-1-like immunoreactivity exists in the renal cortex and medulla of
rats.15 In the heart, ET-1 induces myocardial cell hypertrophy'6 and has positive inotropic'7 and chronotropic'8
effects.
A single subcutaneous injection of monocrotaline
(MCT), a pyrrolizidine alkaloid, causes pulmonary hy-
888
Circulation Research Vol 73, No 5 November 1993
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pertension in rats.'9-21 Pathological changes that occur
in the lungs after MCT administration consist of vascular endothelial cell damage,22 medial thickening of the
muscular pulmonary arteries, and neomuscularization
of nonmuscular distal arteries.20,23 A progressive rise in
pulmonary arterial pressure and the development of
right ventricular (RV) hypertrophy appear to correlate
with these pulmonary vascular structural changes.20'23
Although various studies concerning this animal model
of pulmonary hypertension have been performed, its
etiology is still unknown.
In the present study, the pathophysiological role of
endogenous ET-1 was studied from the viewpoint of its
contribution to the development and/or progression of
cardiopulmonary changes in rats with MCT-induced
pulmonary hypertension (MCT rats). Venous plasma
and lung ET-1 levels, the expression of prepro ET-1
mRNA in various organs, and pulmonary vascular responses to ET-1 were studied in MCT rats at various
disease stages. Furthermore, we studied the effects of
continuous infusion of BQ-123, a novel selective ETA
receptor antagonist,24 on the extent of cardiopulmonary
changes in MCT rats.
Materials and Methods
Animals
Four-week-old male Wistar rats were used. Treatment with MCT (Wako Pure Chemical, Osaka, Japan)
was performed as previously described21; rats were given
a single subcutaneous injection of 60 mg/kg MCT
(MCT rats) or saline (control rats) and were killed after
6, 10, 14, 18, or 25 days.
On the day of the experiment, each rat was artificially
ventilated under anesthesia with sodium pentobarbital
(50 mg/kg IP). The thorax was opened, and a heparinfilled needle, connected to a pressure transducer (model
SCK-590, Gould, Cleveland, Ohio), was inserted into
the RV. RV pressure was recorded by means of a
polygraph system (AP-601G amplifier and WT-687G
thermal pen recorder, Nihon Kohden, Tokyo, Japan). A
blood sample was collected from the RV for determining the plasma ET-1 concentration.
The main pulmonary artery, the aorta, the heart, the
lungs, and the left kidney were removed. The heart was
divided into the RV, left ventricle (LV), septum (SEP),
right atrium (RA), and left atrium (LA), and each
portion was separately weighed and rapidly frozen in
liquid nitrogen. The lungs and the left kidney were
weighed and were also rapidly frozen. The main pulmonary artery and the aorta were immediately placed in
Krebs-Ringer solution.
Sandwich Enzyme Immunoassay to Determine
Plasma and Lung ET-1 Levels
Each blood sample was placed in chilled tubes containing aprotinin (300 kallikrein inhibiting units/mL) and
EDTA (2 mg/mL) and was centrifuged at 2000g for 15
minutes at 4°C. The plasma was stored at -30°C until
used. Plasma (1 mL) was acidified with 3 mL of 4% acetic
acid, and immunoreactive ET-1 was extracted with a
Sep-pak C-18 cartridge (Waters Associates, Milford,
Mass) as previously described.25 The eluates were reconstituted with 0.25 mL of assay buffer and subjected to
sandwich enzyme immunoassay (EIA) for ET-1.
Sandwich EIA for ET-1 was carried out as previously
described using immobilized mouse monoclonal antibody AwETN40, which recognizes the N-terminal portion of ET-1, and peroxidase-labeled rabbit anti-ET-1
C-terminal peptide(15-21)Fab'.25,26 The assay for ET-1
did not cross-react with ET-3 or big ET-1 (crossreactivity, <0.1%). The detection limit of this EIA was
0.4 pg/mL.25,26
The lung ET-1 level was determined as previously
described.27 Briefly, the lung, which was frozen in liquid
nitrogen and stored at -80°C, was homogenized with a
Polytron homogenizer for 60 seconds in 10 vol of 1
mol/L acetic acid containing 10 ,ug/mL pepstatin (Peptide Institute, Osaka, Japan) and immediately boiled for
10 minutes. The homogenate was centrifuged at 20 000g
for 30 minutes at 4°C, and the supernatant was stored at
-30°C until used. The supernatant was subjected to
sandwich EIA for ET-1.
Measurement of the Vascular Response to ET-I
The main pulmonary artery and the aorta from the
rats of both groups were immediately excised and
placed in Krebs-Ringer solution of the following composition (mmol/L): NaCl, 113; KCl, 4.8; MgSO4, 1.2;
CaCl2, 2.2; KH2PO4, 1.2; NaHCO3, 25; and glucose, 5.5.
After the vessel was freed from the surrounding connective tissues, a 4-mm-long vessel ring (ie, pulmonary
artery or aorta) was mounted in a silicon-coated 30-mL
organ bath by means of two metal holders, one being
anchored and the other being connected to a force
displacement transducer (model TB-612T, Nihon Kohden) for the measurement of isometric contractions.
The solution was constantly bubbled with a mixture of
95% 02-5% CO2 and kept at 37°C. The resting tension
was adjusted to 1.0 g. After equilibration for 2 hours,
the response to K' (50 mmol/L) was repeatedly measured at intervals of 30 minutes until a steady response
was obtained (three or four times). The dose-response
relation for ET-1 (Peptide Institute) was determined by
means of cumulative application. The arterial segments
were gently treated so as not to injure the intimal
surface, and the presence of endothelium was confirmed
by the dilator response to acetylcholine (10`6 mol/L).
The cross-sectional area of each pulmonary artery and
aorta was determined from its tissue wet weight and
diameter.28 The contractile tension was divided by the
cross-sectional area for the normalization of the data.
Northern Blot Analysis for Prepro ET-i mRNA
Total RNA from rat tissues (RV, LV, SEP, LA, RA,
lung, kidney) was prepared by selective precipitation in 3
mol/L LiCl and 6 mol/L urea.'3 Five or 10 ,tg per lane of
total RNA prepared from these tissues was separated by
formaldehyde/1.1% agarose gel electrophoresis and
transferred onto nylon membranes (Hybond N, Amersham). The membranes were prehybridized at 42°C for 3
hours in a solution containing 1 mol/L NaCl, 50%
formamide, 1% sodium dodecyl sulfate, and 150 ,g/mL
fragmented salmon sperm DNA and were then hybridized with 32P-labeled cDNA probes in the same solution
at 42°C for 16 hours. The filters were finally washed in
0.1 x standard saline citrate/0.1% sodium dodecyl sulfate
at 50°C and subjected to autoradiography. The prepro
ET-1 cDNA used as a probe in the present study was a
previously described full-length insert of ArET1-213 that
889
Miyauchi et al Endogenous ET-1 in Pulmonary Hypertension
TABLE 1. Hemodynamic Measurements
P<001
p<001
[mmHgl
80i
70-
Day 6
C (n=16)
MCT (n=13)
Day 10
C (n=9)
MCT (n=9)
Day 14
C (n=20)
MCT (n=18)
Day 18
C (n=7)
MCT (n=8)
Day 25
C (n=21)
MCT (n= 16)
60
~
40o.0
30
20
c
10
1.0
0
N. S.
C MCT
Day 6
C MCT
Day 10
C MCT
Day 14
C MCT
Day 18
[m g/mg]
C MCT
Day 25
P
1.0
<o0.oi
P <0.01
Mean BP,
mm Hg
Mean CVP,
mm Hg
101.3±2.3
2.0±0.2
2.4±0.3
111.0±7.5
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111.4±2.8
101.7±5.4
...
110.8±3.3
105.9±4.0
1.7±0.3
1.7±0.2
119.1±6.6
104.4±5.7
2.1±0.4
5.1±0.8*
118.0±4.0
93.3+7.7*
2.1±0.2
7.0±0.7*
BP indicates blood pressure; CVP, central venous pressure;
C, control rats; and MCT, monocrotaline-injected rats. Values are
mean±SEM.
*P<.01 vs the corresponding value in C rats.
RV
is
right ventricle,
LV
is
left ventricle,
and
SEP
is
septum] (B) of control (C) rats and monocrotaline (MOT)-
injected rats on days 6, 10, 14, 18, and 25. Each column
and bar represents the mean±SEM. The number of rats
in each group was the same as that shown in Table 1.
N.S. denotes not significant.
labeled by random priming with [a-32P]dCTP (3000
Ci/mmol, Amersham). To normalize the prepro ET-1
signals for the loaded amounts and transfer efficiencies,
the same membranes were rehybridized with a ,B-actin
cDNA probe as the internal control.
was
Effects of Continuous BQ-123 Infusion on
Pulmonary Hypertension and RV Hypertrophy
and on the Alteration of Lung Vascular
Morphology in MCT Rats
To investigate the effects of continuous BQ-123 infusion on the MCT rats, osmotic minipumps (model 1701,
Alza Corp, Palo Alto, Calif) were subcutaneously implanted in 4-week-old male Wistar rats on the day of
injection of 60 mg/kg MCT or saline. BQ-123 was
dissolved in saline. The content of the osmotic minipumps was saline alone or saline containing BQ-123. The
rats were divided into the following four groups: group I,
MCT rats receiving continuous BQ-123 infusion (7.1 mg
per day per rat) for 18 days; group II, MCT rats receiving
continuous BQ-123 infusion (14.3 mg per day per rat) for
18 days; group III, MCT rats receiving continuous saline
infusion for 18 days; and group IV, control rats receiving
continuous saline infusion for 18 days.
Eighteen days after the injection of MCT or saline,
the rats were killed under pentobarbital anesthesia (50
mg/kg). The hemodynamic parameters were measured,
and the wet weight of each heart was measured.
To histologically investigate the lung, the lungs were
excised and immersed in formalin. Paraffin sections of
4-,um thickness from the middle region of the left and
right lung were stained with hematoxylin and eosin and
were examined under light microscopy.
Statistical analysis of the medial wall thickness was
performed in accordance with the method described by
Ono and Voelkel.29 In brief, measurements of the
external diameter and medial wall thickness were made
on 30 muscular arteries (in the size ranges of 25 to 50, 51
to 100, and 101 to 200 ,um in external diameter) per one
lung section. For each artery, the medial wall thickness
was expressed as follows: % wall thickness= [(medial
thickness x 2)/external diameter] x 100. One lung section
was obtained from one rat. For this analysis, seven
sections were obtained from each group.
Statistics
Data were expressed as mean+SEM. Group means
were compared using an unpaired Student's t test or
one-way analysis of variance with multiple comparisons
where appropriate. A value of P<.05 was accepted as
statistically significant.
Results
Development of Pulmonary Hypertension and RV
Hypertrophy in MCT Rats
A single injection of MCT caused a progressive
increase in RV systolic pressure that was significantly
higher than the control value by day 14, after which it
rose quite rapidly (Fig 1A). MCT also caused a progressive increase in the ratio of RV to LV+SEP from days
14 to 25 that was significantly greater than the control
value, indicating the development of RV hypertrophy in
the MCT rats (Fig 1B). Mean central venous pressure
was significantly elevated on days 18 and 25 in the MCT
rats (Table 1). Mean blood pressure was significantly
lower in the MCT than in the control rats only on day 25
890
Circulation Research Vol 73, No 5 November 1993
TABLE 2. Wet Weight of Hearts and Lungs of Control Rats and Monocrotaline-Injected Rats
(LV+SEP)/BW,
LV+SEP, mg
BW, g
RV, mg
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Day 6
C (n=16)
145.0+4.8
101.5+14.7
356.3+17.0
134.4±4.9
105.7±4.8
MCT (n=13)
332.9+25.7
Day 10
C (n=9)
157.0±5.4
411.1±13.4
128.9±4.2
129.8±4.2*
347.8±20.5t
116.7±5.8
MCT (n=9)
Day 14
C (n=20)
208.1±6.7
492.3+14.4
166.2±4.6
MCT (n=18)
178.3±6.7*
420.0±11.7*
183.1±11.5
Day 18
243.3±4.2
C (n=7)
561.4±16.1
175.7±5.7
MCT (n=8)
185.6±4.5*
486.3+10.2t
325.0±15.5*
Day 25
C (n=21)
291.7±5.3
612.7±13.6
179.2±5.5
MCT (n= 16)
218.8±7.5*
515.0±12.1 *
363.9±15.9*
BW indicates body weight; LV, left ventricle; SEP, septum; RV, right ventricle;
Values are mean±SEM.
*P<.01 and tP<.05 vs the corresponding value in C rats.
(Table 1). The body weight of the MCT rats was
significantly lower than that of the control rats on days
10, 14, 18, and 25 (Table 2). However, the lung wet
weight of the MCT rats was significantly higher than
that of the control rats on days 10, 14, 18, and 25 (Table
2), suggesting the development of inflammatory injury
in the lungs of the MCT rats.
Plasma ET-I Concentrations
The plasma ET-1 concentration of the MCT rats
progressively increased and was significantly higher than
that of the control rats from day 10 (Fig 2). Therefore,
the increase in plasma ET-1 concentration preceded the
development of pulmonary hypertension. On days 18
and 25, the plasma ET-1 concentration of the MCT rats
was markedly elevated (Fig 2).
mg/g
RV/BW,
mg/g
2.69±0.09
2.63±0.07
0.87±0.03
0.85±0.04
1.03±0.09
2.63±0.09
2.68±0.15
0.83±0.03
0.90±0.05
1.33±0.08
1.76±0.16t
2.55±0.08
2.59±0.08
0.86±0.04
1.44±0.11
1.12+0.05*
2.28±0.22*
2.31±0.05
2.63±0.05*
0.72±0.03
1.17±0.05
1.75+0.06*
1.99±0.11*
Lungs, g
1.11+0.14
2.09±0.04
0.61±0.02
1.37±0.06
2.32±0.05*
1.63±0.06*
2.15±0.09*
C, control rats; and MCT, monocrotaline-injected rats.
Vascular Response to ET-1
In the ring preparation of the pulmonary artery, ET-1
produced dose-dependent vasoconstriction in both the
control and MCT rats. In the MCT rats on day 14, ET-1
at low doses (approximately 10-10 to 10-9 mol/L) produced an oscillation-like response (Fig 3B) that was
observed in seven of eight rats. However, this oscillation-like response was not observed in the MCT rats on
either day 6 or day 25 (data not shown). An oscillationlike response was not observed in the control rats at any
stage (ie, on days 6, 14, and 25) (Fig 3A). The doseresponse curves for the vasocontractile response to
ET-1 did not differ significantly between the MCT and
control rats on either day 6 or day 14 (Fig 4, Table 3).
On day 25, both the maximal response and the PD.
value (negative logarithm of concentration producing
[Pg/mI]
10,
P < 0.01
P < 0.01
CO
0
4-a
S.
CO
c
4I)
0
c
0
0
5.
N. S.
P
< 0.01
P
< 0.01
2
to
n
E
0)
'U
*
OJ
t
C MCT
Day 6
m
Se**e
C MCT
Day 10
C MCT.
Day 14
C MCT
Day 18
C MCT
Day 25
FIG 2. Plot shows plasma endothelin-1
(ET-1) concentration in control (C) rats and
monocrotaline (MCT)-injected rats on days
6, 10, 14, 18, and 25. Each point represents
an individual plasma ET-1 concentration
value. Each horizontal line represents the
mean. N.S. denotes not significant.
o
(A) Pulmonary Artery
A Day 141
Day 6
A Control rat
W
I-co.
(911
v
10)
o.-
01
0
891
Miyauchi et al Endogenous ET-1 in Pulmonary Hypertension
c
9.5
8.5
7.5 7Ach
Ac
v
A
KCI
8
Day 25
Day 14
30 min
mm
15 min
c
B MCT rat
0
c
F
(9)
1
757
Wash
v
~~~~~~~~~8.5
v
7
9
Ach
v
v
10 9
8
10 9
7
9.5
KCI min
15
10 9
7
8
7
(B) Aorta
Day 25
~~~~~30 min
Downloaded from http://circres.ahajournals.org/ by guest on June 11, 2017
FIG 3. Typical tracings show the dose-response relation
for the vasocontractile response to endothelin-1 (ET-1) in
pulmonary arteries isolated from control rats (A) and
monocrotaline (MCT)-injected rats (B) on day 14. The
numbers in the figure indicate the antilog molar concentration of ET-1. Ach indicates acetylcholine. ET-1 produced dose-dependent vasocontraction in both groups.
In the MCT rats, but not in the control rats, low-dose ET-1
(3x10-10 and 3x10-9 mol/L) produced an oscillation-like
response.
50% of maximal contraction) were significantly smaller
in the MCT than in the control rats (Fig 4 and Table 3),
indicating that the pulmonary vascular response to ET-1
was significantly reduced in the MCT rats.
In the ring preparation of the aorta, the dose-response curves for the vasocontractile response to ET-1
did not differ significantly between the MCT and control rats on day 25 (Fig 4 and Table 3).
Expression of Prepro ET-i mRNA in Various Tissues
The expression of prepro ET-1 mRNA was altered in
the various tissues of the MCT rats. Typical examples
on days 14 and 25 are shown in Fig 5. Fig 6 depicts
densitometric analysis of this blot corrected for ,3-actin
mRNA, which was used as normalization for a constitutively expressed message.
In the MCT rats, prepro ET-1 mRNA expression in
the lungs was significantly reduced on days 14 and 25
(Figs 5 and 6), whereas that in the kidneys was significantly increased on days 6, 14, and 25 (Fig 6). Prepro
ET-1 mRNA expression in both the RV and LV was
significantly increased on days 18 (Fig 6) and 25 (Figs 5
and 6) in the MCT rats. However, prepro ET-1 mRNA
expression in either RV or LV did not differ significantly between the MCT and the control rats on day 14
(Figs 5 and 6). On day 25, the prepro ET-1 mRNA
expression in SEP, RA, and LA in the MCT rats was
also markedly increased (Fig 5).
Lung ET-i Level
On day 14, the lung ET-1 level of the MCT rats
(3.75+±0.74 ng/g lung) was significantly lower than that
of the control rats (6.47±0.51 ng/g lung, P<.05). On day
25, the lung ET-1 level of the MCT rats (2.69+±0.78 ng/g
lung) was also significantly lower than that of the control
rats (5.74+1.00 ng/g lung, P<.05).
8
ET-1 [-log M]
v
-D 0'5-
0
o- [g/mm2]
Co~
~~~~~
---.--
0.5
MCoTr
@ 05-i
~
~Z
0
O-I
10 9
8
7
ET-1 [-log M]
FIG 4. A, Dose-response curves show the vasocontractile response to endothelin-1 (ET-1) in pulmonary arteries
isolated from control rats and monocrotaline (MCT)injected rats on days 6, 14, and 25. Each circle and bar
represents the mean+SEM of eight samples. The ordinate represents the increase in tension (g/mm2); the
abscissa represents the antilog molar concentration of
ET-1. B, Dose-response curves show the vasocontractile
response to ET-1 in aorta isolated from control rats and
MCT rats on day 25. Each circle and bar represents the
mean+SEM of seven samples. The ordinate represents
the increase in tension (glmm2); the abscissa represents
the antilog molar concentration of ET-1.
c
Effects of Continuous BQ-123 Infusion on
Pulmonary Hypertension, RV Hypertrophy,
and the Alteration of Lung Vascular
Morphology in MCT Rats
Continuous infusion of BQ-123 (7.1 mg per day per
rat) for 18 days significantly reduced RV systolic pressure (Fig 7) and significantly ameliorated RV hypertrophy (Fig 7 and Table 4). Continuous infusion of BQ-123
at a higher dose (14.3 mg per day per rat) for 18 days
was more effective at ameliorating both pulmonary
hypertension and RV hypertrophy (Fig 7 and Table 4).
Histology of the lungs of the MCT rats revealed an
increase in pulmonary arterial medial thickness (Figs
8B and 9). Continuous infusion of BQ-123 (14.3 mg per
day per rat) significantly inhibited the observed increase
in the medial thickness of the pulmonary arteries of the
MCT rats (Figs 8C and 9).
Discussion
showed
The present study
that, after treatment with
MCT, pulmonary hypertension in rats developed significantly by day 14 and markedly after day 18. An
important finding was that the plasma ET-1 level also
progressively increased. This increase preceded the
development of pulmonary hypertension, suggesting
Circulation Research Vol 73, No 5 November 1993
892
TABLE 3. PD2 Value and Maximum Response to Endothelin-1 in Pulmonary Artery
and Aorta
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Pulmonary artery
Day 6
Max response, g/mm2
PD2, -log ED50
Day 14
Max response, g/mm2
PD2, -log ED50
Day 25
Max response, g/mm2
PD2, -log ED50
Aorta
Day 25
Max response, g/mm2
PD2, - log ED
Control
MCT
P
0.84±0.09
8.88+0.08
0.86±0.04
8.69+0.07
NS
NS
0.91 + 0.05
8.98±0.03
0.79+0.09
9.04±0.07
NS
NS
0.65+0.03
8.71 ±0.07
0.27+0.02
8.37±0.06
<.01
<.01
NS
NS
MCT indicates monocrotaline-injected rats; PD2, negative logarithm of concentration producing
50% of maximal contraction; and NS, not significant. Values are mean-+-SEM.
0.50±0.01
8.76±0.05
that ET-1 may be involved in the pathogenesis of
pulmonary hypertension in the MCT rats. The elevated
plasma ET-1 level did not originate in the lungs, however, since both the peptide level of ET-1 and the
expression of prepro ET-1 mRNA in the lungs were
significantly lower in the MCT than in the control rats at
0.45±0.02
8.69±0.09
all stages of pulmonary hypertension. The expression of
prepro ET-1 mRNA was already significantly elevated
on day 6, ie, in the prehypertensive stage, only in the
kidneys. Therefore, the increased plasma ET-1 level
seen at the prehypertensive stage in the MCT rats may
be partly attributed to increased ET-1 production in the
14 days after monocrotaline (MCT) injection
LV
C MCT
RV
C
MCT
SEP
C MCT
C
LA
MCT
RA
C
MCT
LUNG
C MCT
prepro ET-1
(2.3 Kb)
I
*93
+4j3Actin
25 days after monocrotaline (MCT) injection
LV
RV
SEP
LA
RA
CMcr
C M CT
C MCT
CMcT
C MCT
.
.
*
0.
.
I, US
I, I, ?1
LUNG
C MCT
4 prepro ET-1
(2.3 Kb)
3,-Actin
FIG 5. Typical examples of Northern blot analysis
are shown for the levels of prepro endothelin-1
(ET-1) mRNA in the heart and the lungs of control
(C) rats and monocrotaline (MCT)-injected rats on
day 14 (top) and day 25 (bottom). To normalize the
prepro ET-1 signals for the loaded amounts and
transfer efficiencies, f3-actin mRNA levels were compared as the internal control. The prepro ET-1
mRNA (2.3 kb) and ,B-actin mRNA (2.0 kb) are
indicated by arrows. LV indicates left ventricle; RV,
right ventricle; SEP, septum; LA, left atria; and RA,
right atria. Ten micrograms per lane of total RNA
prepared from these tissues, except for the LA and
RA of rats on day 14, was loaded. Five micrograms
per lane of total RNA from the LA and RA of rats on
day 14 was loaded. On day 14, the expression of
prepro ET-1 mRNA in the LV, RV, SEP, LA, and RA
of the MCT rats was not different from that of the C
rats, whereas the expression of prepro ET-1 mRNA
in the lungs of the MCT rats was decreased. On day
25, the expression of prepro ET-1 mRNA in the LV,
RV, SEP, LA, and RA of the MCT rats was markedly
increased, whereas that in the lungs was decreased.
Miyauchi et al Endogenous ET-1 in Pulmonary Hypertension
1
Lung
Kidney
I
893
P <0.05
1.0
N.S.
1.51
P <005 P <0.5
0.51
P<0.05
N.S.
7
11
05
a:
c
E IZ
a
, E
da
C MCT C MCT
Day 6 Day14
i
C MCT
Day 25
LiiZ1
.1P
(D
E
C MCT C MCT C MCT
Day 6 DaylO Day 14
0
F
2
Ll
N.S.
L----- .
M
N.S.
Downloaded from http://circres.ahajournals.org/ by guest on June 11, 2017
Day 6 Day14 Day18 Day25
Day14 Day18 Day25
Day 6
FIG 6. Bar graphs show the level of expression of prepro endothelin-1 (ET-1) mRNA in various tissues from control (C)
rats and monocrotaline (MCT)-injected rats. The autoradiogram from Northern blot analysis was scanned by a
densitometer, and the ratio between the levels of prepro ET-1 mRNA and f-actin mRNA was calculated. The data for lung,
kidney, right ventricle (RV), and left ventricle (LV) of the C rats and MCT rats at various stages are shown. Each column
and bar represents the mean+±SEM of three samples. N.S. denotes not significant. In this experiment, the lung samples
from all stages were transferred onto one membrane. The kidney samples from all stages were also transferred onto a
membrane. The RV and LV samples were transferred onto respective membranes. Therefore, the typical examples of
Northern blot analysis shown in Fig 5 do not accurately represent the mean of multiple animals shown in this figure.
kidneys. Since MCT causes nephrotoxicity,30 the increase in prepro ET-1 mRNA expression in the kidneys
might be due to a direct action of MCT on the kidneys.
The expression of prepro ET-1 mRNA in both the RV
and LV in the MCT rats significantly increased at the
severe pulmonary hypertensive stage, ie, on days 18 and
25, suggesting that the marked increase in plasma ET-1
observed at this stage may be partly ascribed to the
increase in ET-1 production in the heart as well as in the
kidneys. Since circulating ET-1 is mainly eliminated by
uptake into the lungs and the kidneys,3' a decrease in
ET-1 elimination by the lungs due to inflammatory
injury of the lung may also contribute to the marked
increase in plasma ET-1.
Although the dose-response curves for ET-1 in the
pulmonary artery did not differ between the control and
0
c
[mmHg]
[mg/mgI] I]
1.0
100
I
~~P<0.01
P<0.01 P<0.01
a.
CL
0
1
cn)
4.,
o
c) 50
> 0.5
1-
N-
._
0
13
4.'
0
0
4-i
CD
if
C
MCT MCT MCT
+
+
+
+
Saline Saline BO-123 BO-123
(7.1ymg/)
md/r
day/rat (14.3
day/rat
4
0
C
MCT MCT MCT
+
+
+
+
Saline Saline BO-123 BO-123
f 7.1 mg/) ( 14.3 m4/l
vday/rat s day/rat .1
FIG 7. Bar graphs show the effects of continuous infusion of BQ-1 23, a selective ETA receptor antagonist, on the
progression of pulmonary hypertension (left) and right ventricular (RV) hypertrophy (right) in the monocrotaline
(MCT)-injected rats. C+saline indicates control rats given continuous saline infusion for 18 days (n=8); MCT+saline,
MCT rats given continuous saline infusion for 18 days (n=10); MCT+BQ-123 (7.1 mg/day/rat), MCT rats given
continuous BQ-123 infusion (7.1 mg per day per rat) for 18 days (n=9); MCT+BQ-123 (14.3 mg/day/rat), MCT rats given
continuous BQ-1 23 infusion (14.3 mg per day per rat) for 18 days (n=7); LV, left ventricle; and SEP, septum. Each column
and bar represents the mean and SEM.
894
Circulation Research Vol 73, No 5 November 1993
TABLE 4. Body Weight and Wet Weight of Hearts of Control Rats and Monocrotaline-Injected Rats Given Continuous
Infusion of BQ-123 or Saline
C+Saline (n=8)
MCT+saline (n=10)
MCT+ BQ-1 23
7.1 mg per day per rat (n=9)
14.3 mg per day per rat (n=7)
279.6+20.1
(LV+SEP)/BW,
mg/g
2.18+0.03
2.18-+0.05
RV/BW,
mg/g
0.68+0.01t
1.23+0.09
232.2+10.9*
2.29±0.04
219.7±13.0*
2.30+0.07
1.01 +0.05*
0.95+0.05*
BW, g
2519+3.3*
LV+SEP, mg
548.0+11.5*
170.8-4.1t
227.8+5.8
496.5+13.7
236.9+6.7
231.9+4.1
544.3+18.3
534.7+21.3
RV, mg
BW indicates body weight; LV, left ventricle; SEP, septum; RV, right ventricle; C+saline, control rats given continuous saline infusion
for 18 days; MCT+saline, monocrotaline (MCT)-injected rats given continuous saline infusion for 18 days; and MCT+BQ-1 23, MCT rats
given continuous BQ-123 infusion for 18 days. Values are mean-+-SEM.
*P<.05 and tP<.01 vs the corresponding value for MCT+saline.
Downloaded from http://circres.ahajournals.org/ by guest on June 11, 2017
MCT rats on either day 6 or day 14, ET-1 at low doses
produced an oscillation-like response only in the MCT
rats on day 14, suggesting that the vascular responsiveness
to ET-1 was altered at this stage. Nevertheless, the pulmonary artery exhibited a significantly reduced response
to ET-1 in the MCT rats on day 25. Since the pulmonary
vasocontractile response to prostaglandin F1,')2 or 5-hydroxytryptamine33 has been shown to be increased in
MCT rats at the pulmonary hypertensive stage, the reduction in the response to ET-1 seems to be a phenomenon
relatively specific for ET-1. In the present study, MCT did
not affect the vasocontractile response to ET-1 in the
aorta, indicating that the reduction in the vascular response to ET-1 occurred specifically in the pulmonary
artery. ET-1 activates vascular ETA receptors, thereby
causing vasoconstriction via several intracellular signal
transduction systems.34
The ETA receptor can easily be downregulated by
exposure to endothelins.35 MCT has been reported to
rBr
injure the endothelium of pulmonary arteries but not of
the aorta. MCT-induced endothelial injury causes an
increase in pulmonary vascular permeability and the
exudation of plasma components to the pulmonary
vascular wall but not to the aortic wall.30,36 Therefore, it
is possible that elevated circulating ET-1 in MCT rats
can easily penetrate into the pulmonary vessel wall but
probably not into the aortic wall. This phenomenon may
have led to the selective downregulation of ETA receptors, thereby reducing the response to ET-1 in the
pulmonary artery. Lerman et a137 have reported that
acute systemic administration of ET-1 producing a
twofold increase in its circulating concentration results
in significant cardiovascular and renal biological activity, ie, decreased heart rate and cardiac output in
association with increased renal and systemic vascular
resistance in dogs. In the present study, however, systemic blood pressure did not significantly change (Table
1) despite a fivefold increase in plasma ET-1 concentra-
FIG 8. Photomicrographs show the effects of continuous
BQ-123 infusion on the lung vascular morphology of
monocrotaline (MCT)-injected rats. A, Control rats were
given continuous saline infusion for 18 days. B, MCT rats
were given continuous saline infusion for 18 days. C,
MCT rats were given continuous BQ-123 infusion (14.3
mg per day per rat) for 18 days. Each arrow indicates the
section of the pulmonary artery; Br, bronchus. Lung
histology showed a marked increase in pulmonary arterial medial thickness in the MCT rats (B). Continuous
infusion of BQ-123 significantly inhibited the increase in
the medial thickness of the pulmonary arteries of the MCT
rats (C). Bar= 100 pm.
Miyauchi et al Endogenous ET-1 in Pulmonary Hypertension
p
<0.01
p
<0.01
p
<o0.01 p<0.01
t 20-
0
p <o.o1
p
<o.oi
c
0
I- 10.30
o-
C
MCT MCT
+
+
Saline Saline BO-123
25 -. .Sm
+
MCT MCT
C
+
+
Saline Saline BO-123
51 -100 0m
|
+
MCT MCT
C
+
+
+
Saline Saline BO-123
101-200m
External Diameter
Downloaded from http://circres.ahajournals.org/ by guest on June 11, 2017
tion in MCT rats on day 18. The decrease rather than
increase in systemic blood pressure on day 25 may have
been due to a continuous severe sickness (see the body
weight in Table 2). Therefore, it is likely that circulating
ET-1 plays a relatively selective role in the alteration of
the cardiopulmonary system of MCT rats. Although the
exact reason for the selective reduction in the pulmonary vascular response to ET-1 is unclear, this finding
suggests that excessive stimulation of ETA receptors
selectively occurs in the pulmonary vascular smooth
muscles of the MCT rats.
In the present study, the ETA receptor antagonist
BQ-123 significantly inhibited the development of pulmonary hypertension and RV hypertrophy. Since ET-1 is
a potent pulmonary vasoconstrictor,5-7 one possible
mechanism for the inhibition of pulmonary hypertension
by BQ-123 may be antagonism of the vasocontractile
action of endogenous ET-1. Indeed, ET-1 induced an
oscillation-like response in the pulmonary artery of the
MCT rats on day 14. However, since the pulmonary
vascular response to ET-1 was markedly reduced in the
MCT rats on day 25, it is unlikely that the inhibition of
sustained pulmonary hypertension by BQ-123 was solely
due to the antagonism of ET-1-induced vasoconstriction.
Increased thickness of the pulmonary arterial media is
believed to play a major role in the development of
MCT-induced pulmonary hypertension and sustained
irreversible increases in pulmonary vascular resistance.202338 ET-1 has potent proliferative activity on
vascular smooth muscle cells.49 The present histological
examination revealed that BQ-123 effectively prevented
arterial medial thickening in the MCT rats, suggesting
that endogenous ET-1 participates in the thickening of
pulmonary vessels. These findings are in agreement with
the demonstration that ET-1-induced DNA synthesis is
inhibited by BQ-123 in cultured rat vascular smooth
muscle cells.39 Therefore, it is conceivable that the inhibition of pulmonary hypertension by BQ-123 is, at least
in part, due to the antagonism of ET-1-induced vascular
proliferation. This assumption is consistent with the
above-mentioned argument that the excessive stimula-
895
FIG 9. Bar graphs show the effects of continuous BQ-123 infusion on the percent wall
thickness of the pulmonary artery of monocrotaline (MCT)-injected rats. C+saline indicates control rats given continuous saline
infusion for 18 days; MCT+saline, MCT rats
given continuous saline infusion for 18 days;
and MCT+BQ-123, MCT rats given continuous BQ-123 infusion (14.3 mg per day per
rat) for 18 days. Measurements of the external diameter and medial wall thickness were
made on 30 muscular arteries (in the size
ranges of 25 to 50, 51 to 100, and 101 to 200
,um in external diameter) per one lung section. For each artery, the medial wall thickness was expressed as follows: % wall
thickness= [(medial thickness x 2)/external
diameter]xl00. One lung section was obtained from one rat. Each column and bar
represents the mean±SEM of seven rats.
tion of ETA receptors may selectively occur in the pulvascular smooth muscle of the MCT rats.
RV hypertrophy occurred concomitantly with the
development of pulmonary hypertension after MCT
treatment; it was moderate on day 14 and marked after
day 18. Another important finding in the present study
was the dramatic increase in prepro ET-1 mRNA
expression in the hearts of the MCT rats at the severe
pulmonary hypertensive stage (after day 18). The increase in prepro ET-1 mRNA expression in the heart
may have been due to overload of the heart caused by
severe pulmonary hypertension. It has been demonstrated that the 5' flanking region of the prepro ET-1
gene has three octanucleotide sequences that conform
with a consensus of AP-1/Jun-binding elements and
that phorbol esters actually activate prepro ET-1
mRNA expression in cultured endothelial cells.40 Furthermore, pressure overload to the heart has been
shown to cause an increase in c-fos and c-jun expression
in the early stage.41 Therefore, it is likely that pressure
overload to the heart may induce prepro ET-1 mRNA
expression via the expression of the trans-acting transcription factors Fos and Jun. Since ET-1 was shown to
induce myocardial cell hypertrophy,16 the marked increase in ET-1 production in the heart may also contribute to an exaggeration of cardiac hypertrophy after
day 18. We believe that the inhibition of RV hypertrophy by BQ-123 can be partly attributed to the blockade
of excessive stimulation of the heart by ET-1, in addition
to the prevention of pulmonary hypertension.
It has been reported that lung ET-1 production
increased in rats with idiopathic pulmonary hypertension despite a lack of increase in plasma ET-1.42 In
contrast, lung prepro ET-1 mRNA expression in rats
with pulmonary hypertension due to chronic hypoxia
does not differ from that in control rats.42 Thus, lung
ET-1 production appears to differ among animal models
of pulmonary hypertension. In the present study, lung
ET-1 production decreased in the MCT rats. As a
possible mechanism for this, it can be speculated that
severe inflammation of the lung parenchyma or severe
damage of the pulmonary endothelial cells caused by
monary
896
Circulation Research Vol 73, No 5 November 1993
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MCT might result in a reduction of ET-1 production in
the lung. The presence of injured vascular endothelial
cells in the pulmonary circulation, but not in the
systemic circulation, has been shown in patients with
pulmonary hypertension.43 Furthermore, increased circulating levels of ET-1 have been demonstrated in
patients with both primary and secondary forms of
pulmonary hypertension.44'45 These phenomena are
similar to those seen in the MCT rats.
It has been reported that platelet-activating factor
antagonists inhibit MCT-induced lung injury and pulmonary hypertension.29 In addition, p-chlorophenylamine, a
5-hydroxytryptamine synthesis inhibitor, has also been
shown to inhibit MCT-induced pulmonary hypertension
in rats.4' Thus, MCT-induced pulmonary hypertension
appears to be caused by several etiologic factors. In the
present study, we demonstrated that an ETA receptor
antagonist inhibited the development of pulmonary hypertension, suggesting that endogenous ET-1 probably
contributes to the progression of cardiopulmonary alterations in MCT rats. Although the precise mechanism of
the inhibitory effects of an ETA receptor antagonist is
unclear, the present findings may provide new directions
for analyzing the mechanisms of progression of pulmonary hypertension and for developing beneficial treatments for pulmonary hypertension.
Acknowledgments
This study was supported by a grant from the Special
Research Project on the Circulation Biosystem from the
University of Tsukuba and a grant from the Study Group of
Molecular Cardiology. We would like to thank Dr Yukio
Fujimaki and Dr Masahiko Kobayashi for useful discussions
concerning the histological examination portion of our study.
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Downloaded from http://circres.ahajournals.org/ by guest on June 11, 2017
Contribution of endogenous endothelin-1 to the progression of cardiopulmonary
alterations in rats with monocrotaline-induced pulmonary hypertension.
T Miyauchi, R Yorikane, S Sakai, T Sakurai, M Okada, M Nishikibe, M Yano, I Yamaguchi, Y
Sugishita and K Goto
Circ Res. 1993;73:887-897
doi: 10.1161/01.RES.73.5.887
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