Department of Pulmonary Pharmacology, UW2532, SmithKline Beecham
Pharmaceuticals, King of Prussia, Pennsylvania
This study investigated the effects of the nonpeptide endothelin (ET)
receptor antagonist, SB 217242, against ET-1-induced pulmonary pressor
responses and in a model of hypoxia-induced pulmonary hypertension in
the guinea pig. In guinea pig isolated pulmonary artery rings, SB
217242 (3-300 nM) produced a concentration-dependent inhibition of
ET-1-induced contractions, with a pA2 of 8.1. SB 217242 (1 or 3 mg/kg i.v.) elicited a dose-related inhibition of ET-1-induced
increases in pulmonary artery and airway insufflation pressure
responses in anesthetized guinea pigs. Chronic exposure to hypoxia (9%
O2 for 0-14 days) produced a time-dependent increase in
mean pulmonary artery pressure. After a 10-day exposure to hypoxia
there was about a 100% elevation in pulmonary artery pressure, and
right ventricular mass and plasma irET levels increased 3-fold compared
with normoxic animals. SB 217242, administered by continuous intraperitoneal infusion via mini osmotic pump (0.36, 3.6 or 10.8 mg/day), significantly reduced (by about 50%)
hypoxia-induced pulmonary artery pressure increases at all three doses
used. The hypoxia-induced right ventricular hypertrophy was
significantly attenuated by the 3.6 and 10.8 mg/day doses. Based on
hematocrit, hemoglobin and red blood cell counts, SB 217242 did not
affect the normal physiological erythropoietic response to hypoxia.
There were no appreciable differences in the maximum contractile
effects of ET-1 or the potency of SB 217242 (pKB values,
8.3 and 8.0, respectively) versus ET-1-induced responses
in isolated pulmonary arteries from hypoxic versus
normoxic guinea pigs. However, there was a marked reduction in
endothelium-dependent relaxation of precontracted pulmonary artery
isolated from hypoxic compared with normoxic animals. The results of
the present study provide further preclinical evidence for a
pathophysiological role of ET-1 and the potential therapeutic utility
of ET receptor antagonists, such as SB 217242, in pulmonary
hypertension.
 |
Introduction |
Pulmonary
hypertension is a major complication of chronic hypoxia that may result
from chronic obstructive pulmonary disease, congestive heart failure,
respiratory distress syndrome, cystic fibrosis and hypoventilation
syndrome (Zapol and Snider, 1977
; Cody et al., 1992
; MacNee,
1994). Right ventricular hypertrophy, increased vascular resistance and
enhanced vascular remodeling, including hyperplasia (increase in
myocardial and smooth muscle cell number), hypertrophy (increase in
muscle cell size) and muscle extension (appearance of new smooth muscle
in previously less muscularized arterioles), are hallmarks of chronic
hypoxia in the pulmonary circulation, which combine to produce an
anatomic resistance to flow (Reid, 1979
). Several animal models of
hypoxia-induced pulmonary hypertension have been developed, primarily
in the rat, and these have been used to investigate the mechanisms
underlying the vascular constriction and remodeling components of the
disease (Grover et al., 1963
; Rabinovitch et al.,
1979
; Thompson et al., 1989
). In our laboratory, a model of
hypoxia-induced pulmonary hypertension in an optimized environment for
the guinea pig has been developed and characterized (Bochnowicz
et al., 1997
).
ET-1, originally identified in 1988 (Yanagisawa et al.,
1988
), is a member of a family of 21-amino-acid peptides, which
includes ET-2 (two amino acid substitution from ET-1) and ET-3 (six
amino acid substitution) (Inoue et al., 1989
; Masaki
et al., 1992
). The ETs produce an array of effects in many
biological systems, which are mediated via two G
protein-coupled, seven-transmembrane-spanning receptors, designated
ETA and ETB (Masaki
et al., 1992
; Sakurai et al., 1990
). In the lung
ET-1 has been implicated in the pathophysiology of several diseases,
including pulmonary hypertension (Stewart et al., 1991
;
Giaid et al., 1993
; Hay et al., 1993a
, b; Hay and Goldie, 1995
; Michael and Markewitz, 1996
). The postulated relationship to pulmonary hypertension is based primarily on the following observations: 1) ET-1 is a potent constrictor of mammalian pulmonary blood vessels, including human pulmonary artery and vein (Brink et al., 1991
; Hay et al., 1993a
, b); 2) ET-1 is a
mitogen for human pulmonary artery smooth muscle cells via
an ETA receptor-mediated mechanism (Zamora
et al., 1993
); 3) there are numerous reports of increased
plasma levels and enhanced expression of ET in individuals with
pulmonary hypertension (Cernacek and Stewart, 1989
; Stewart et
al., 1991
; Yoshibayashi et al., 1991
; Allen et
al., 1993
; Giaid et al., 1993
). In addition, in rat
models of pulmonary hypertension, ET receptor antagonists have
attenuated the characteristic functional and morphological changes
(Bonvallet et al., 1994
; Chen et al., 1995
;
DiCarlo et al., 1995
; Oparil et al., 1995
).
During the past few years several peptide and nonpeptide ET receptor
antagonists have been identified (Warner et al., 1996
). SB
217242 is a recently identified nonpeptide compound which is a highly
potent antagonist for ETA
(Ki for displacement of
[125I]ET-1 = 1.1 nM) and to a lesser
extent ETB receptors
(Ki = 111 nM) (Ohlstein et al.,
1996
). The compound has good oral bioavailability (67% in the rat
after intraduodenal infusion) and, thus, has the appropriate
pharmacodynamic profile to be a useful tool to assess the potential
pathophysiological role of ET-1 in diseases at the preclinical level.
The purpose of the present study was to characterize the effects of SB
217242 on exogenous ET-1-induced pressor responses in the pulmonary
vasculature and in a chronic hypoxia-induced model of pulmonary
hypertension in the guinea pig.
 |
Methods |
Animals.
Male Hartley guinea pigs (Charles River, Portage,
MI; weight range, 600-800 g) were randomly selected and assigned to
one of seven groups: 1) normoxic, no treatment, n = 6;
2) hypoxic, vehicle, n = 7; 3) hypoxic, SB 217242, 0.36 mg/day, n = 7; 3) hypoxic, SB 217242, 3.6 mg/day,
n = 5; 4) hypoxic, SB 217242, 10.8 mg/day,
n = 5; 5) normoxic, vehicle, n = 5; and
6) normoxic, SB 217242, 10.8 mg/day, n = 6. In the
text, animals exposed to conditions of hypoxia (9%
O2) or normoxia (18% O2)
are referred to as "normoxic" or "hypoxic" guinea pigs.
Similarly, pulmonary artery preparations from the two sets of animals
are indicated in some places in the text as "normoxic" or
"hypoxic" tissues or preparations.
Guinea pig isolated pulmonary artery studies.
The pulmonary
artery was dissected from lungs of male Hartley guinea pigs and
positioned around a 21 G blunted syringe needle. Each tissue was
cleaned of adherent material and then cut into four 2-mm-wide rings.
Individual rings were placed in 10-ml organ baths containing modified
Krebs-Henseleit solution gassed with 95% 02, 5%
CO2 and maintained at 37°C; the pH was 7.4. The
composition of the Krebs-Henseleit solution was (mM): NaCl, 113.0; KCl,
4.8; CaCl2, 2.5;
KH2PO4, 1.2;
MgSO4, 1.2; NaHCO3, 25.0;
and glucose, 5.5. Preparations were connected via stainless
steel hooks and silk suture to Grass FTO3C force-displacement
transducers and mechanical responses were recorded isometrically by
MP100WS/Acknowledge data acquisition system (BIOPAC Systems, Goleta,
CA) run on Macintosh computers. Tissues were equilibrated under
approximately 1.5 g resting load, based on previous preliminary
data (Hay et al., 1993b
), for at least 1 hr before the start
of each experiment; during this period, preparations were washed every
15 min with fresh Krebs-Henseleit solution. After the equilibration
period, tissues were precontracted with 100 mM KCl. The tissues were
then rinsed every 15 min for about 1 hr to return the level of tone to
base-line values. The preparations were then left for at least 30 min
before the start of the experiment. ET-1 concentration-response curves
were obtained by its cumulative addition to the organ bath in half-log
increments (Van Rossum, 1963
). Each drug concentration was left in
contact with the preparation until the response reached a plateau
before addition of the subsequent agonist concentration. At the end of
the experiment, tissues were exposed again to 100 mM KCl, which served
as a reference contraction for data analysis. Paired tissues were
exposed to SB 217242 (3-300 nM) or saline vehicle for 30 min before
ET-1 cumulative concentration-response curves were initiated. Only one
agonist concentration-response curve was generated per tissue.
In some experiments the endothelium-dependent relaxation induced by
carbachol was compared in pulmonary arteries isolated from normoxic and
hypoxic animals. For these studies, pulmonary artery rings with intact
endothelium were contracted with 100 mM KCl, and after plateau of this
response, tissues were exposed to cumulative additions of carbachol,
administered in half-log increments. After completion of carbachol
concentration-response curves, each tissue was given papaverine (100 µM), which produces the maximum relaxation and was used as the
reference response for data analysis.
Osmotic pump implantation.
On day 0, animals were placed
individually into a 6-liter chamber and anesthetized with isoflurane
gas for intraperitoneal implantation of mini osmotic pumps, model 2 ML2
(Alzet, Palo Alto, CA), after surgical procedures outlined in the Alzet
technical information manual. Sterile conditions were maintained to
reduce the incidence of infection associated with surgery, and surgical instruments were sterilized between procedures on each animal with a
hot bead sterilizer (Inotech Biosystems, Lansing, MI). The drug was
solubilized in sterile solution and osmotic pumps were filled with
either sterile water or SB 217242 immediately before surgery. The
implantation site was shaved and cleaned first with alcohol and
Betadine solution, and the animals were placed in a head box to
maintain anesthesia. A midline incision, 1 to 2 cm long, was made in
the lower abdomen posterior to the rib cage. The musculoperitoneal
layer was carefully pulled up to avoid damage to the bowel, and the
layer directly beneath the incision of the cutaneous layer was incised.
A filled osmotic pump was then inserted, delivery portal first, into
the peritoneal cavity. The musculoperitoneal layer was closed with a
running 3-0 silk suture, and the cutaneous incision closed with two to
four wound clips. Animals were removed from the head box and returned
to room air to recover (for 10 min). Computer-readable tags (BioMedic Data Systems, Maywood, NJ) were injected subcutaneously into the scruff
of the neck to allow for accurate identification and to monitor weight
and behavioral changes of the individual animals throughout the
experiment. Animals were allowed to recover for a minimum of 1 hr, then
placed into the hypoxic chamber. SB 217242 or vehicle is released from
the osmotic pumps immediately after implantation and begins operating
at a constant rate, 5.0 µl/hr, within 4 to 6 hr, for a maximum
duration of 14 days.
Hypoxia chamber.
The chambers were designed by us to provide
an optimized environment for hypoxic exposure of guinea pigs
(Bochnowicz et al., 1997
) and custom made (Mitchell
Plastics, Norton, OH) to meet USDA and SmithKline Beecham requirements
in accordance with the Animal Welfare Act. Animals were housed for
various times, up to a maximum of 14 days, in sealed 212-liter acrylic
chambers. The hypoxia chamber was designed to house eight animals,
exceeding the USDA requirement of 103 square inches of flooring/animal
weighing more than 350 g, and totaling a floor area of 864 square
inches. The chamber was designed with two swing-down, sealed doors on the lower front panel to allow access to the pans for cleaning on a
daily basis without changing the entire cage, thus reducing the time of
exposure to a normoxic environment. Chambers were cleaned and
disinfected every third day while the animals were being weighed.
Hypoxia (9% O2) was produced by mixing equal
flow rates of compressed air and nitrogen gas. Gases were circulated in
the chamber with a fan (12 V DC, 3.5 inch square), and gas samples were
taken twice daily with a Fyrite O2 and
CO2 analyzer (Baccarach, Pittsburg, PA). On
returning the animals to the chamber, a 90-min equilibration period
occurred before 9% O2 was reached inside the
chamber. Food and water were provided ad libitum. Special feeders and a continuous watering system were constructed to eliminate typical spilling and emptying of water bottles and food cups. Note,
that "normoxic" animals were not placed in these chanbers. Previous
studies indicated that there were was no difference between the
physiological parameters in normoxic chamber-housed versus conventionally housed guinea pigs (data not shown).
Acute ET-1-induced pulmonary pressor and bronchoconstrictor
responses.
Male Hartley guinea pigs (600-800 g) were anesthetized
with sodium pentobarbital (40 mg/kg i.p.), and both external jugular veins, the carotid artery and the trachea were cannulated for drug
administration, blood and airway pressure monitoring and ventilation.
The pulmonary artery was cannulated and pressure was measured as
outlined below. Bilateral vagotomies were performed to minimize neural
reflex influences. The animals were paralyzed with pancuronium bromide
(0.1 mg/kg i.v.) and ventilated at 45 breaths/min. Airway pressure
changes were measured via a side arm of the tracheal cannula
with a Druck PDCR 10/2L, 70 mBar transducer (Druck Incorporated, New
Raifield, CT). The ventilatory stroke volume of a Harvard rodent
respirator (model 683) was set to produce a side-arm pressure of 8 cm
of H2O (~5 cc room air). Blood pressure was
measured with a Kobe model CDXIII disposable pressure transducer (Kent
Scientific, Litchfield, CT). Saline vehicle or SB 217242 (1 or 3 mg/kg
i.v.) was administered 5 min before ascending doses of ET-1 (0.01-3
µg/kg, i.v.) via the left external jugular vein, and changes in
pulmonary artery and airway pressure were monitored. Pressures were
recorded on a thermal chart recorder (Model WR 3300, Western Graphtec;
Irvine, CA).
Pulmonary artery pressure measurement.
On day 0, 3, 7, 10 or
14 (day 10 for antagonist studies), animals were removed from the
chamber and anesthetized with 40 mg/kg i.p. sodium pentobarbital.
Animals were allowed to breath spontaneously. A small incision was made
on the center line at the neck to expose the carotid artery, which was
cannulated for blood collection with a 19 GA 8-inch Intracath Vialon
catheter (Hanna's Pharmaceutical Supply, Wilmington, DE) for chemistry
and hematology analysis. The catheter was then connected via
a Kobe disposable pressure transducer (Kent Scientific, Litchfield, CT)
to a chart recorder (Western Graphtec WR 3300) for monitoring systemic
arterial pressure. To measure pulmonary artery pressure, a
custom-formed 3.5 French Argyl umbilical catheter was introduced into
the right external jugular vein and advanced through the atrium and
right ventricle into the pulmonary artery. The catheter was then
connected via a pressure transducer to the chart recorder
and monitored for 10 min. The position of the catheter was determined
by the waveform of the pressure tracing and confirmed upon necropsy.
Histology.
After the period of monitoring pulmonary artery
pressure, the animals were sacrificed by an overdose of sodium
pentobarbital. A midline incision was made along the length of the
whole body on the front side, and the animals were exsanguinated by
severing the inferior vena cava. The heart, lungs and trachea were
removed en bloc and the heart dissected free for immediate
gravimetric analysis. The vasculature and atrial appendages were
removed from the heart and the right ventricle wall dissected free and
weighed. The resulting weights were reported as the weight ratio of the right free wall to body weight to provide an index of right ventricular hypertrophy.
Blood chemistry and hematology and plasma concentrations of SB
217242.
Blood (1 ml) was drawn before pulmonary artery
catheterization and split into two samples: one 400-µl sample was
transferred to a Microtainer tube containing lithium heparin for blood
chemistry, and one 500-µl sample was transferred to a Microtainer
tube containing ethylenediaminetetraacetic acid for hematological
analysis. Blood samples were analyzed by the Clinical Unit of our
Laboratory Animal Sciences Department on a Monarch 2000 clinical
chemistry analyzer (Instrumentation Laboratories, Lexington, MA) and a
Technicon H 1.E hematology analyzer (Tarrytown, NY). Immediately after
the period of monitoring pulmonary arterial pressure, 3 ml of blood was
drawn into a heparinized syringe, divided into two Eppendorf tubes and
spun in a microcentrifuge (Eppendorf model 5415 C) at 12,000 rpm for 12 min. Plasma was collected into a single Eppendorf tube and frozen for
ET-1 and SB 217242 analysis. Plasma levels of ET-1 were determined by
endothelin immunoassay which is 100% specific for ET-1, ET-2, ET-3 and
sarafotoxin, but has less than 0.01% specificity for big ET, based on
a double-antibody "sandwich" technique (minimum detectable
concentration
1.5 pg/ml; Cayman Chemicals, Ann Arbor, MI).
Concentrations are expressed as picograms per milliliter of plasma.
Plasma concentrations of SB 217242 were determined by protein
precipitation and quantified by liquid chromatography/tandem mass
spectrometry. The lower limit of quantitation was 10.0 ng/ml based on a
50-µl plasma sample.
Data analysis.
All data were reported as mean ± S.E.M.
For the results of the in vivo studies, statistical analyses
were performed using a factorial measures ANOVA; significance was
determined with the Fisher's PLSD at 95% or a 99% level of
confidence (Statview II, Abacus Concepts Inc., Berkley, CA). For
in vitro contraction studies, agonist-induced responses for
each tissue were expressed as a percentage of the reference carbachol
(10 µM)-induced contraction obtained at the end of the experiment
("postcarbachol"). Geometric mean EC50 values
(pD2 values) were calculated from linear
regression analyses of data. Where appropriate, antagonist potencies
were calculated and expressed as pKB and
pA2; pKB =
log
[antagonist]/X
1, where X is the ratio
of agonist concentration required to elicit 50% of the maximal
contraction in the presence of the antagonist compared with that in its
absence and pA2 =
log of the antagonist dissociation constant. Results for control and treated tissues were
analyzed for differences in both the EC50 values
and the maximum contractile responses. Statistical analysis was
conducted by ANOVA or two-tailed Student's t test for
paired samples where appropriate with a probability value less than .05 regarded as significant.
Drugs.
The following drugs were used: ET-1 (human, porcine)
was obtained from Peninsula Laboratories (Belmont, CA). Carbachol and papaverine was purchased from Sigma Chemical Co. (St. Louis, MO). SB
217242 was synthesized by colleagues in the Department of Medicinal Chemistry, SmithKline Beecham Pharmaceuticals (King of Prussia, PA).
 |
Results |
Guinea pig isolated pulmonary artery studies.
In pulmonary
artery rings isolated from normoxic guinea pigs, ET-1 (0.1 nM to 1 µM) potently produced concentration-related contractions with a
pD2 of 8.31 ± 0.03 (n = 5).
The maximum response elicited by ET-1 (0.3 µM) represented
approximately 75% of that induced by 100 mM KCl (fig.
1). SB 217242 (3-300 nM) produced a
concentration-dependent inhibition of ET-1-induced contractions, reflected by marked shifts to the right in agonist
concentration-response curves; Schild plot analysis of the data
revealed a pA2 of 8.1, with a slope that was not
significantly different from 1, which indicated competitive antagonism.
SB 217242 did not have an effect on the response induced by ET-1 or
KCl, the reference contraction. (P = .64, ANOVA, fig. 1).

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Fig. 1.
Effects of SB 217242 (3-300 nM) against ET-1
concentration-response curves in guinea pig pulmonary artery ring
preparations (endothelium denuded). Results are expressed as a
percentage of the response to KCl (100 mM) added at the end of the
experiment, and are given as the mean ± S.E.M.;
n = 4-5.
|
|
There were no differences in the potency of ET-1 in pulmonary arteries
from normoxic and hypoxic guinea pigs (10-day exposure, 9%
O2) [pD2, 8.04 (normoxic)
and 8.09 (hypoxic), n = 5]. However, there was a small
decrease (P = .049) in the maximum response produced by ET-1 in
pulmonary artery preparations from normoxic (76.5 ± 4.0%)
versus hypoxic (60.2 ± 5.7%) guinea pigs (data
expressed as % of KCl (100 mM)-induced maximum contraction;
n = 5; fig. 2A). In
addition, the potency of SB 217242 (3 µM) for inhibition of
ET-1-induced contractions was slightly different in tissues taken from
normoxic and hypoxic animals, with respective pKB
values of 7.9 and 8.3, P = .048; n = 5) (fig. 2A).
In contrast, the endothelium-dependent relaxation elicited
by carbachol was markedly reduced in pulmonary artery from normoxic
versus hypoxic guinea pigs (P = .013; fig. 2B). The
maximum relaxation induced by 1 µM carbachol (% 100 µM papaverine)
was: normoxic tissues, 57.6 ± 8.9% (n = 5);
hypoxic tissues, 20.3 ± 6.7%, n = 5. Note, the
maximal contractions induced by KCl (100 mM) (P = .76) and maximal
relaxation elicited by papaverine (100 µM) (P = .17) were
similar in normoxic and hypoxic tissues.

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Fig. 2.
Comparison of (A) effects of SB 217242 against
ET-1-induced contractions and (B) relaxant responses to carbachol in
pulmonary artery obtained from normoxic and hypoxic (10-day exposure to 9% O2) guinea pigs. Results are expressed as a percentage
of the response to (A) KCl (100 mM) or (B) papaverine (100 µM) added at the end of the experiment, and are given as the mean ± S.E.M. (A) n = 5.
|
|
Exogenous ET-1 in anesthetized guinea pigs.
In anesthetized,
paralyzed and ventilated guinea pigs, ET-1 (0.01-3 µg/kg i.v.)
produced dose-related increases in pulmonary artery pressure (fig.
3A) and airway insufflation pressure
(fig. 3B); the maximum response for both parameters occurred at a dose of 1 µg/kg i.v. The maximum increase in pulmonary vascular pressure was 11.6 ± 1.6 mm Hg (n = 5), which represented a
77% increase from resting mean pulmonary artery pressure. Base-line
pulmonary artery pressure, measured immediately before the first dose
of ET-1, was not different when comparing vehicle-treated (15 ± 0.8 mm Hg; n = 5) or SB 217242 (1 or 3 mg/kg
i.v.)-treated guinea pigs (16 + 0.9 and 16.4 + 0.4 mm Hg, respectively;
n = 5) (fig. 3A).

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Fig. 3.
Effects of SB 217242 (1 or 3 mg/kg i.v.; 5-min
pretreatment) versus ET-1 (0.01-3 µg/kg i.v.)-induced
(A) pressor responses and (B) bronchoconstriction in anesthetized,
paralyzed, ventilated guinea pigs. Results are expressed as percent
change from base line and are given as mean ± S.E.M.
*Significantly different from vehicle-treated control animals;
n = 5.
|
|
Vehicle or SB 217242 did not alter the base-line airway insufflation
pressure (data not shown). ET-1 (1 µg/kg i.v.) produced a maximal
increase in airway insufflation pressure of 15.8 ± 3.2 cm
H2O which represented approximately a 200%
increase from base line.
Pretreatment with SB 217242 (1 or 3 mg/kg i.v. 5 min before agonist
administration) produced a dose-related inhibition of ET-1-induced
increases in pulmonary artery pressure and airway insufflation pressure
responses, manifest by significant rightward shifts in the
dose-response curves for each parameter (fig. 3, A and B).
Chronic hypoxia studies.
Chronic exposure to hypoxia (9%
O2 for 0-14 days) produced a time-dependent
increase in mean pulmonary artery pressure which reached statistical
significance on the 7th day of hypoxia when compared with day 0 (fig.
4; ANOVA, Fisher's PLSD;
n = 4-6). The pulmonary artery pressure approximately
doubled from 14.3 ± 0.9 and 13.4 ± 0.4 mm Hg on day 0 in
normoxic untreated animals and normoxic vehicle-treated animals,
respectively, to a maximum of 26.8 ± 0.6 mm Hg on day 10 in
hypoxic, vehicle-treated animals (fig. 4). Based on this finding, the
10-day time point was selected for the drug comparator studies. In
addition, in vehicle-treated/hypoxia-exposed guinea pigs, right
ventricular mass, expressed as a percentage of body weight, was
significantly greater when compared with normoxic animals (fig.
5). However, histological analysis
(hematoxylin and eosin and elastin staining) revealed no obvious
evidence of pulmonary vascular remodeling in the various regions of the
guinea pig lung that were examined.

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Fig. 4.
Effects of exposure to hypoxia (9% O2
for 0-14 days) on pulmonary artery pressure in guinea pigs. Results
are expressed as mm Hg and are given as mean ± S.E.M.
*Significantly different from day zero, n = 4-6.
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|

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Fig. 5.
Effects of SB 217242 on pulmonary artery pressure
in normoxic and 10-day hypoxic guinea pigs. Vehicle or SB 217242 (3, 30 or 90 mg/ml) were delivered i.p. by osmotic pump at 5 µl/hr. Results are expressed as mm Hg and are given as mean + S.E.M. *Significantly different from vehicle-treated hypoxic animals; n = 5-7.
|
|
Administration of SB 217242 (0.36, 3.6 or 10.8 mg/day) for 10 days, by
continuous infusion via mini osmotic pump, resulted in a
significant reduction (about 50%) in hypoxia-induced pulmonary artery
pressure increases, measured on day 10, at all three doses used (ANOVA,
Fisher's PLSD, n = 5-7, fig. 5). The hypoxia-induced right ventricular hypertrophy also was abrogated significantly by the
3.6 and 10.8 mg/day doses, but not by the 0.36 mg/day dose (ANOVA,
Fisher's PLSD, n = 4-6; fig.
6).

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Fig. 6.
Effects of SB 217242 on hypoxia-induced right
ventricular hypertrophy in guinea pigs. Weight of free wall of the
right ventricle as a percentage of body weight in either normoxic or
10-day hypoxic guinea pigs. Vehicle or SB 217242 (3, 30 or 90 mg/ml)
were delivered i.p. by osmotic pump at 5 µl/hr. * Significantly
different from vehicle-treated animals; n = 5-7.
|
|
Immunoreactive ET concentrations in plasma were significantly increased
(approximately 3-fold) in 10-day hypoxic guinea pigs (35.4 ± 7.7 pg/ml; n = 6) when compared with normoxic animals (12.7 ± 1.8 pg/ml; n = 4; P < .05, ANOVA,
Fisher's PLSD) (table 1). ET-1 levels in
hypoxic animals which were treated with SB 217242 at the 0.36 mg/day
dose (9.5 ± 4.6 pg/ml; n = 6) and the 3.6 mg/day
dose (5.1 ± 0.9 pg/ml; n = 5) were significantly
lower than those of hypoxic vehicle-treated guinea pigs (P < .005, respectively; ANOVA, Fisher's PLSD) and no different from
concentrations in normoxic animals (P > .05, ANOVA) (table 1).
Plasma levels in the SB 217242 10.8 mg/day hypoxia-exposed group
(40.6 ± 7.1 pg/ml; n = 8) were significantly
greater than the normoxic vehicle group (P < .005) and the
normoxic SB 217242 10.8 mg/day group (9.5 ± 3.5 pg/ml;
n = 8; P < .005), but not different from the
hypoxic vehicle group (P > .05) (table 1).
Plasma concentrations of SB 217242 were not detectable
(i.e., less than 10 ng/ml) in the 0.36 mg/day treatment
group or the vehicle-treatment group. In the 3.6 mg/day hypoxia
treatment group, plasma concentrations were 57.8 ± 44.4 ng/ml. In
the 10.8 mg/day hypoxic group, the plasma concentration of SB 217242 was 270.6 ± 49.6 ng/ml, which was slightly greater than the
149.3 ± 28.2 ng/ml (P = .207, not significant) found in the
corresponding normoxic 10.8 mg/day treatment group.
The 10-day hypoxia in vehicle-treated guinea pigs resulted in
significant increases in hematocrit (39%), hemoglobin (30%) and red
blood cell concentrations (27%) in whole blood compared with
corresponding normoxic animals (P < .05; ANOVA and Fisher's PLSD; table 2). In hypoxic animals
treated with any of the three doses of SB 217242, hematological
parameters were not significantly different from vehicle-treated
hypoxic animals (P > .05; table 2). In addition, hematocrit,
hemoglobin and red blood cell concentrations in normoxic guinea pigs
treated with the highest dose of SB 217242 (10.8 mg/day) were not
different from normoxic animals (P > .05; table 2).
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TABLE 2
Effects of SB 217242 on hematology in normal and hypoxic guinea
pigsa
Results are expressed as mean ± S.E.M.
|
|
 |
Discussion |
The major findings of the present study are: 1) exposure of guinea
pigs to hypoxia (9% O2, 10 days) produced an
increase in pulmonary artery pressure, right ventricular hypertrophy
and an elevation in irET plasma levels; 2) SB 217242, a potent and
selective nonpeptide ET receptor antagonist, inhibited exogenous
ET-1-induced elevation in pulmonary artery pressure and airway
insufflation pressure, and the hypoxia-induced increases in pulmonary
artery pressure, right ventricular hypertrophy and irET plasma levels; 3) hypoxia had little or no influence on the sensitivity of isolated pulmonary artery preparations to ET-1 and the potency of SB 217242 against ET-1-induced contractions, but caused a marked reduction in the
endothelium-dependent relaxation compared with that observed in tissues
obtained from normoxic guinea pigs; and 4) the normal hematological
response to chronic hypoxia (i.e., increased hematocrit and
hemoglobin resulting from increased red blood cell synthesis and
release) were not affected by SB 217242.
The results of the present study clearly describe the efficacy of the
nonpeptide endothelin receptor antagonist, SB 217242, against
hypoxia-induced pulmonary hypertension and right ventricular hypertrophy in the guinea pig. The present protocol has been previously well described as an optimal, animal-friendly and relevant model of
hypoxia that consistently produces pulmonary vascular and right ventricular pathological sequelae which mimic the changes seen in
pulmonary hypertension resulting from hypoxia produced in a variety of
clinical conditions (Bochnowicz et al., 1997
). The endothelin receptor antagonist, SB 217242, is an orally bioavailable compound which preferentially antagonizes ETA
receptors (Ki = 1.1 nM) compared with
ETB receptors (Ki = 111 nM) (Ohlstein et al., 1996
).
Two seven-transmembrane-spanning G protein-coupled ET receptors, termed
ETA and ETB, have been
cloned with human tissue (Arai et al., 1993
; Hosoda et
al., 1992
). ET-1 and ET-2 have significantly higher affinity than
ET-3 for ETA receptors. ETB
receptors, which recognize the identical carboxy-terminal ends of the
ETs, bind all three ligands with similar affinity (Sakurai et
al., 1990
; Arai et al., 1990
; Masaki et al.,
1992
). Both receptor subtypes are present in mammalian lung, and their
activation has been demonstrated to produce many effects in this system
including bronchoconstriction, vascular smooth muscle contraction,
microvascular permeability, mucus secretion, smooth muscle and
fibroblast proliferation, inflammatory cell activation and modulation
of neurotransmission (Hay et al., 1993a
; Hay and Goldie,
1995
; Michael and Markewitz, 1996
). In guinea pig pulmonary artery,
contractions induced by ET-1 were sensitive to BQ-123 and were proposed
to be mediated by ETA receptor activation (Hay
et al., 1993b
). This was confirmed in the present study, in
which SB 217242 produced a concentration-dependent antagonism of
ET-1-induced contractions.
The ETB receptor has been termed a "clearance
receptor" based initially on studies in the rat where
ETB receptors appear to mediate lung clearance of
ETs (Fukuroda et al., 1994
; Sato et al., 1995
).
Further evidence supporting this postulate includes the finding of a
doubling of plasma ET-1 levels in humans treated with the combined
ETA/ETB receptor
antagonist, bosentan (Kiowski et al., 1995
). Although some
controversy exists concerning ET plasma levels in pulmonary
hypertension and hypoxia, the overall evidence points to an increase in
ET synthesis and release in relevant clinical conditions and animal
models (Michael and Markewitz, 1996
). The plasma concentrations of irET
in the present study were elevated (about 3-fold) with chronic 10-day
hypoxic exposure in vehicle-treated animals when compared with normoxic
animals. In the two lower-dose SB 217242-treated animals, irET
concentrations were significantly lower than corresponding
vehicle-treated animals and not different from normoxic animals. This
finding might suggest that increased plasma ET levels were the
result and not the cause of hypoxic pulmonary
hypertension. Plasma levels of irET in hypoxic guinea pigs treated with
the highest dose of SB 217242 were nearly the same as corresponding
vehicle-treated hypoxic animals. A plausible explanation might be a
reduction in clearance of ET associated with antagonism of the
ETB receptor subtype at the highest dose of SB
217242. The demonstration that plasma levels in normoxic animals
treated with the highest dose of SB 217242 were no different from
normoxic animals suggests that SB 217242 does not, per se, raise base-line irET plasma concentrations. These findings suggest that
plasma concentrations of ET in normoxic animals are in an equilibrium
of formation and catabolism by peptidase activity and/or excretion.
When hypoxia exists, plasma ET levels increase because of increased
release from the endothelium. Although SB 217242 is significantly more
selective for the ETA subtype (100-fold vs. ETB), at higher doses, antagonism
of the ETB receptor (the so-called "clearance
receptor") may occur. Although this receptor may not be important
when ET release is low (i.e., normoxic states), it may play
a more important role in hypoxic states in which excessive ET release
may saturate catabolic peptidase activity which is usually able to
handle normal ET levels to maintain its equilibrium.
The present finding of a reduced relaxant effect of carbachol in
precontracted pulmonary artery from hypoxic guinea pigs compared with
normoxic animals further highlights the potential pathophysiological significance associated with hypoxia. Nitric oxide, the molecular species which mediates the endothelial-dependent relaxant effects of
various vasorelaxants, including carbachol, may play a role in both ET
synthesis and activity (Ryan et al., 1993
; Kourembanas et al., 1993
; Markewitz et al., 1995
). Patients
with primary pulmonary hypertension have decreased expression of
constitutive nitric oxide synthase which may contribute to enhanced ET
expression (Giaid et al., 1993
; Giaid and Saleh, 1995
). The
physiological effects of ET-1 are modulated by nitric oxide through: 1)
decreasing ET-1 release from endothelial cells (Kourembanas et
al., 1993
), 2) attenuating ETA receptor
affinity and 3) blunting ET-1-induced increases in intracellular
calcium (Goligorsky et al., 1994
). Therefore, decreased
nitric oxide synthetic capacity, demonstrated by the present in
vitro results, which indicate decreased endothelium-dependent relaxation, could lead to enhanced ET formation and release. A loss of
endothelium-dependent relaxant activity in a perfused lung preparation
has been demonstrated in rats exposed to hypoxia (Adnot et
al., 1991
) and in porcine pulmonary artery, but not vein, exposed
in vitro to acute hypoxia (Félétou et
al., 1995
). Maximum contractile activity to ET-1 in pulmonary
artery from hypoxic guinea pigs was similar to that in preparations
from normoxic animals. Furthermore, the sensitivity
(pD2 values) of tissues from normoxic or hypoxic
animals were not significantly different, which suggests no appreciable
ETA receptor desensitization, and the lack of
difference in KCl-induced maximal contractions in normoxic
versus hypoxic animals, which suggests no difference in
muscular potential for contraction. In addition, there appeared to be a
very small difference (P = .48 based on pKB
values) in in vitro potency of SB 217242 in pulmonary artery
from hypoxic versus normoxic animals.
A role for endothelin in hypoxia-induced pulmonary vascular and right
heart remodeling has been suggested by studies in which the
ETA receptor antagonist, BQ-123, was shown to
prevent and reverse these changes in the rat (Bonvallet et
al., 1994
; Chen et al., 1995
; DiCarlo et
al., 1995
; Oparil et al., 1995
). The reduction, but not
total prevention, in the present study with SB 217242 may result from
differences in causative factors in a particular species, subtle
differences in the models or intrinsic differences in the compounds
used. In addition, chronic hypoxic exposure in rats resulted in
increased ET-1 synthesis and enhanced ETA and
ETB receptor mRNA, which correlated with
increased pulmonary artery pressure and right ventricular hypertrophy
(Elton et al., 1992
; Li et al., 1994a
, b).
Several groups have shown an increase in circulating levels of ET-1 in
patients with pulmonary hypertension (Cernacek and Stewart, 1989
;
Stewart et al., 1991
; Yoshibayashi et al., 1991
;
Allen et al., 1993
). Whether a true "cause and effect relationship" exists between endothelin and enhanced pulmonary vasoreactivity and vascular remodeling resulting from hypoxia is not
known. However, all of this evidence clearly implicates a consistent
association.
The lack of the effect of SB 217242 on the hypoxia-induced increases in
hematocrit and hemoglobin has two major implications: 1) that
pharmacological antagonism of ET receptors by SB 217242 would not
inhibit important erythropoietic survival mechanisms and, therefore,
not contribute to tissue pathology resulting from hypoxic insult or
high-altitude exposure, and 2) that ET receptors do not modulate
hypoxia-induced erythropoietin release from the kidney.
In summary, the present results in a guinea pig model of
hypoxia-induced pulmonary hypertension provide additional evidence supporting a significant role of ET in the pathophysiology of pulmonary
hypertension and the potential utility of potent and selective ET
receptor antagonists, such as SB 217242, in the treatment of this
condition, by attenuating its two major features, the increase in
pulmonary artery pressure and the morphological changes in the heart.
The authors thank the staff of the Department of Laboratory
Animal Sciences at SmithKline Beecham Pharmaceuticals for their assistance in animal care and diagnostics.
Accepted for publication August 5, 1997.
Received for publication October 31, 1996.