Mechanism of hypoxic pulmonary vasoconstriction involves
ETA receptor-mediated inhibition of KATP
channel
Koichi
Sato,
Yoshiteru
Morio,
Kenneth G.
Morris,
David
M.
Rodman, and
Ivan F.
McMurtry
Cardiovascular Pulmonary Research Laboratory, Department of
Medicine, University of Colorado Health Sciences Center, Denver,
Colorado 80262
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ABSTRACT |
There is controversy on the role of endothelin (ET)-1 in
the mechanism of hypoxic pulmonary vasoconstriction (HPV). Although HPV
is inhibited by ET-1 subtype A (ETA)-receptor antagonists in animals, it has been reported that ETA-receptor blockade
does not affect HPV in isolated lungs. Thus we reassessed the role of
ET-1 in HPV in both rats and isolated blood- and physiological salt
solution (PSS)-perfused rat lungs. In rats, the
ETA-receptor antagonist BQ-123 and the nonselective
ETA- and ETB-receptor antagonist PD-145065, but
not the ETB-receptor antagonist BQ-788, inhibited HPV.
Similarly, BQ-123, but not BQ-788, attenuated HPV in blood-perfused lungs. In PSS-perfused lungs, either BQ-123, BQ-788, or the combination of both attenuated HPV equally. Inhibition of HPV by combined BQ-123
and BQ-788 in PSS-perfused lungs was prevented by costimulation with
angiotensin II. The ATP-sensitive K+
(KATP)-channel blocker glibenclamide also prevented
inhibition of HPV by BQ-123 in both lungs and rats. These results
suggest that ET-1 contributes to HPV in both isolated lungs and intact animals through ETA receptor-mediated suppression of
KATP-channel activity.
adenosine 5'-triphosphate-sensitive potassium channel; hypoxia; endothelin-1; endothelin-receptor blockers; glibenclamide; pulmonary vascular regulation
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INTRODUCTION |
THE MECHANISM OF ACUTE HYPOXIC pulmonary
vasoconstriction (HPV) has not been fully defined. It is believed that
hypoxia acts either directly on peripheral pulmonary arterial smooth
muscle cells to inhibit a voltage-sensitive K+ channel and
cause membrane depolarization, Ca2+ influx through L-type
Ca2+ channels, and contraction (2, 31, 32, 47) or
indirectly to stimulate release of vasoconstrictors and/or inhibit
release of vasodilators (10, 30, 42). Various endothelium-derived vasoactive factors have been proposed to modulate HPV.
Endothelin (ET)-1 is a potent vasoactive agent that is produced by
numerous cell types including pulmonary vascular endothelial cells
(23). Two ET-1 receptor subtypes, ETA and ETB,
have been identified in the pulmonary vasculature. Both ETA
and ETB receptors are on vascular smooth muscle cells and
mediate vasoconstriction (13, 22, 36). ETB receptors are
also on endothelial cells and cause vasodilation through release of
nitric oxide and prostacyclin (8, 14, 20, 27, 36). There is controversy
about the role of ET-1 in the mechanism of HPV. Although in vivo
studies show that HPV is markedly attenuated by either selective
ETA- or nonselective ETA- and
ETB-receptor antagonists (4, 5, 9, 30, 41, 46),
ETA-receptor blockade has been reported not to inhibit HPV
in isolated lungs (17, 40). This apparent disparity suggests that
either ETA-receptor blockade has effects in vivo that are
not mimicked in vitro or the mechanism of HPV differs between the two preparations.
Thus the purpose of this study was to reassess the role of ET-1 in the
mechanism of HPV both in vivo and in vitro. We examined the effects of
ET-1 antagonists on HPV in conscious catheterized rats and isolated rat
lungs perfused with either blood or physiological salt solution (PSS).
Moreover, because increased activity of ATP-sensitive K+ (KATP) channels inhibits HPV (3, 7,
11, 29, 45), because ET-1 suppresses KATP-channel
activity through activation of ETA receptors (18, 26, 43),
and because it has been speculated that HPV is due to ET-1- dependent
inhibition of KATP channels (1), we tested whether the
KATP-channel blocker glibenclamide prevented inhibition of
HPV by ETA-receptor blockade. The results in rats confirmed
that a selective ETA- and nonselective ETA- and
ETB-receptor antagonists but not a selective
ETB-receptor antagonist blocked HPV. HPV was also
attenuated only by the ETA-receptor antagonist in isolated
blood-perfused rat lungs. In contrast, however, either an
ETA- or an ETB-receptor antagonist attenuated HPV in PSS-perfused rat lungs. Furthermore, the inhibition of HPV by
ETA-receptor blockade was prevented by pretreatment with glibenclamide in both lungs and rats. These results suggest that ET-1
participates in the mechanism of HPV in both isolated lungs and intact
animals through an ETA receptor-mediated inhibition of
KATP channels.
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METHODS |
Conscious catheterized rats. The first experiment examined the
effects of ET-1-receptor antagonists on HPV in catheterized rats. Adult
male Sprague-Dawley rats (250-350 g) were anesthetized with
ketamine (100 mg/kg im) and rompun (15 mg/kg im), and catheters were
implanted in the jugular vein and pulmonary and right carotid arteries
as previously described (29). Two days later, conscious rats were
placed in a small ventilated plastic box, and pulmonary and systemic
arterial pressures were measured with pressure transducers. Cardiac
output was determined by a standard dye-dilution method, and total
pulmonary and systemic resistances were calculated by dividing mean
arterial pressure by cardiac output. The plastic box was flushed
continuously with room air except during exposure of the rats to acute
hypoxia when it was flushed with a gas mixture of 10%
O2-90% N2.
Isolated perfused lungs. The second series of experiments
examined the effects of ET-1-receptor antagonists on HPV in isolated blood- and PSS-perfused rat lungs. The lungs were isolated from adult
male Sprague-Dawley rats (250-350 g) after anesthesia with intraperitoneal pentobarbital sodium (30 mg) and an intracardiac injection of 100 IU of heparin. Isolated lungs were ventilated with a
humid mixture of 21% O2-5% CO2-74%
N2 at 60 breaths/min, an inspiratory pressure of 9 cmH2O, and an end-expiratory pressure of 2.5 cmH2O. They were perfused through a main pulmonary arterial cannula with a peristaltic pump at a constant flow of 0.04 ml · g body
wt
1 · min
1.
Effluent perfusate drained from a left ventricular cannula into a
perfusate reservoir. Mean perfusion pressure was measured continuously with a transducer and pen recorder. Blood-perfused lungs were perfused
in a recirculating manner with 20 ml of heparinized whole blood
obtained from methoxyflurane-anesthetized blood donor rats. PSS-perfused lungs were perfused with Earle's balanced salt solution (Sigma) containing Ficoll (4 g/100 ml, type 70; Sigma) as a colloid and
3.1 µM sodium meclofenamate (Sigma) to inhibit synthesis of vasodilator prostaglandins and enhance hypoxic pressor responsiveness. After the lungs were flushed of blood with 20 ml of PSS, they were
perfused with a recirculating volume of 30 ml. Blood- and PSS-perfused
lungs were equilibrated for 20 min at 38°C before hypoxic pressor
responses were elicited. Because the lungs were perfused at a constant
flow and ventilated at constant pressures, changes in perfusion
pressure reflected changes in vascular resistance.
Experimental protocols. To confirm previously reported
inhibition of HPV by ETA-receptor blockade in intact
animals (4, 5, 9, 30, 41, 46), we first examined the effects of ET-1
antagonists on HPV in conscious catheterized rats. The rats were
pretreated with the selective ETA-receptor antagonist
BQ-123 (50 µg · kg
1 · min
1
iv; Banyu Pharmaceutical) (15) or vehicle (0.9% saline; 24 µl · kg
1 · min
1
iv) for 45 min before exposure to two successive hypoxic challenges (10% O2 for 5-6 min at 15-min intervals). Preliminary
experiments showed that this dose of BQ-123 did not affect the initial
transient systemic depressor response to an intravenous bolus of ET-1
(0.22 µg/kg; decrease in mean systemic pressure was
18 ± 4 mmHg in BQ-123-treated rats vs.
25 ± 4 mmHg in control rats;
n = 3 each) but did reduce the secondary sustained systemic
vasoconstriction (increase in total systemic resistance was 278 ± 48 mmHg · l
1 · min
in BQ-123-treated rats vs. 1,312 ± 50 mmHg · l
1 · min
in control rats; n = 3; P = 0.055). To test the effects of the nonselective ETA- and ETB-receptor
antagonist PD-145065 (13, 44) and the selective
ETB-receptor antagonist BQ-788 (16), the rats were first
challenged with hypoxia (10% O2 for 5-6 min) and then
treated with PD-145065 (100 µg · kg
1 · min
1
iv; Parke-Davis), BQ-788 (50 µg · kg
1 · min
1
iv; Banyu Pharmaceutical), or vehicle [24
µl · kg
1 · min
1
iv; saline for PD-145065; dimethyl sulfoxide (DMSO; Sigma) for BQ-788] for 45 min before a second exposure to hypoxia.
Preliminary experiments showed that this dose of PD-145065 inhibited
both the initial systemic depressor response (decrease in mean systemic pressure was
0.4 ± 0.4 mmHg in blocker-treated rats vs.
33 ± 4 mmHg in control rats; P < 0.05; n = 7 and 9, respectively) and the secondary systemic vasoconstriction
(increase in total systemic resistance was 78 ± 74 mmHg · l
1 · min
in blocker-treated rats vs. 771 ± 207 mmHg · l
1 · min
in control rats; P < 0.05) to the intravenous bolus of ET-1
(0.22 µg/kg). In contrast, BQ-788 inhibited the initial depressor response (decrease in mean systemic pressure was
1 ± 1 mmHg in BQ-788-treated rats vs.
30 ± 4 mmHg in control rats; P < 0.05; n = 4 and 5, respectively) but not the secondary
pressor response (increase in mean systemic arterial pressure was
21 ± 7 vs. 23 ± 2 mmHg) to intravenous ET-1.
We next examined effects of selective ETA- and
ETB-receptor antagonists on HPV in blood-perfused lungs.
After two challenges with hypoxic ventilation (0% O2 for
10 min at 10-min intervals) to elicit hypoxic pressor responses, the
lungs were challenged successively with 5, 3, and 0%
O2-5% CO2-balance N2 gas mixtures for 10 min each at 10-min intervals. After the last hypoxic response, either BQ-123 (5 µM) plus DMSO (0.05%; vehicle for BQ-788), BQ-788 (5 µM) plus 0.9% saline (vehicle for BQ-123), or vehicle was added to the perfusate, and the lungs were again challenged with 5, 3, and
0% O2. This experiment showed that HPV was attenuated by the ETA- but not by the ETB-receptor antagonist
in blood-perfused lungs.
We next examined the effects of ET-1 antagonists on HPV in PSS-perfused
lungs. After two initial hypoxic challenges (0% O2 for 10 min at 10-min intervals), the lungs were challenged successively with
the 5, 3, and 0% O2 gas mixtures for 10 min each at 10-min intervals. Either BQ-123 (5 µM) plus DMSO, BQ-788 (5 µM) plus saline, the combination of BQ-123 and BQ-788, or the vehicle was then
added to the perfusate, and the lungs were again challenged with 5, 3, and 0% O2. After completion of the hypoxic challenges, 10 nM ET-1 (human ET-1, Peptide Institute) was added to the perfusate, and
the change in perfusion pressure was measured 10 min later to test for
ET-1-receptor blockade.
Because either BQ-123 or BQ-788 alone or a combination of both
attenuated HPV in PSS-perfused lungs and because it has been reported
that ETA-receptor blockers do not inhibit HPV in
PSS-perfused rat lungs costimulated with angiotensin II (17, 40), we
tested the effect of combined BQ-123 and BQ-788 on HPV in PSS-perfused lungs challenged alternately with angiotensin II and hypoxia. The
protocol was the same as in the preceding experiment except that
angiotensin II (0.3 µg, human, acetate salt; Sigma) was injected as a
bolus into the pulmonary arterial cannula 5 min before each hypoxic
challenge (5, 3, and 0% O2). After the last hypoxic
challenge after the addition of BQ-123 and BQ-788, 10 nM ET-1 was added to the perfusate to test for ET-1-receptor blockade.
To test whether the inhibition of HPV by ETA-receptor
blockade was associated with activation of KATP channels
(18, 26, 43), we examined the effects of BQ-123 on HPV in both PSS- and blood-perfused lungs pretreated with the KATP-channel
blocker glibenclamide. After two initial hypoxic challenges (0%
O2 for 10 min at 10-min intervals), glibenclamide (Sigma)
or vehicle (0.05% DMSO) was added to perfusate. The concentrations of
glibenclamide were 20 µM in PSS-perfused lungs and 50 µM in
blood-perfused lungs and were determined in preliminary experiments to
inhibit the vasodilator response to the K+-channel
activator cromakalim (5 µM; Sigma) during an ongoing hypoxic pressor
response (data not shown). After either glibenclamide or vehicle, the
lungs were again challenged twice with hypoxia (0% O2)
before the addition of either BQ-123 (5 µM) or vehicle (saline) to
the perfusate and two more challenges with hypoxia.
To examine the effects of glibenclamide in catheterized rats, three
groups were studied: rats treated with the vehicle for glibenclamide
and then BQ-123, rats treated with glibenclamide and then BQ-123, and
rats treated with glibenclamide and then the vehicle for BQ-123. The
rats were challenged with hypoxia (10% O2 for 5-6
min) and then treated with either glibenclamide (20 mg/kg iv) or
vehicle (0.5 ml DMSO/kg iv) 45 min before a second hypoxic challenge.
BQ-123 (50 µg · kg
1 · min
1
iv) or vehicle (24 µl
saline · kg
1 · min
1
iv) was then infused for 45 min before a final hypoxic exposure. In
other rats, this dose of glibenclamide blocked cromakalim (20 µg
iv)-induced systemic vasodilation (data not shown).
Statistics. Data are expressed as means ± SE. Statistical
analysis was done by Student's t-test or two-way analysis of
variance, with Fisher's post hoc test for multiple comparisons.
Differences were considered significant at P < 0.05.
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RESULTS |
Effects of ET-1-receptor antagonists on HPV in intact rats.
There were no significant differences in baseline (normoxic) systemic and pulmonary hemodynamics between control and BQ-123-treated rats
(Table 1). Whereas acute hypoxia increased
pulmonary arterial pressure and total pulmonary resistance, i.e.,
caused HPV, it had little or no effect on the other hemodynamic
parameters. HPV was markedly attenuated in the BQ-123-treated rats
(Table 1, Fig. 1). Similarly, the
nonselective ETA- and ETB-receptor blocker PD-145065 did not affect systemic or pulmonary hemodynamics during normoxia (Table 2) but blocked HPV (Table
2, Fig. 2). In contrast, the selective
ETB-receptor antagonist BQ-788 had no effect on HPV (Table
3, Fig. 3).

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Fig. 1.
Selective endothelin type A (ETA) receptor antagonist
BQ-123 inhibited hypoxic pulmonary vasoconstriction in rats. Rats were
pretreated for 45 min with either vehicle (control; n = 7) or
BQ-123 (50 µg · kg 1 · min 1
iv; n = 6) before 2 hypoxic challenges (5-6 min of 10%
O2) were given 15 min apart. TPR, hypoxia-induced
increase in total pulmonary resistance. Values are means ± SE.
* Significant difference from respective control value, P < 0.05 by ANOVA.
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Fig. 2.
Nonselective ETA- and ETB-receptor antagonist
PD-145065 inhibited hypoxic pulmonary vasoconstriction in rats. Hypoxic
challenges (5-6 min of 10% O2) were given before and
45 min after treatment with either vehicle or PD-145065 (100 µg · kg 1 · min 1
iv). Values are means ± SE; n = 7 rats/group.
* Significant difference from respective control value, P < 0.05 by ANOVA.
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Fig. 3.
Selective ETB-receptor antagonist BQ-788 had no effect on
hypoxic pulmonary vasoconstriction in rats. Hypoxic challenges
(5-6 min of 10% O2) were given before and 45 min
after treatment with either vehicle or BQ-788 (50 µg · kg 1 · min 1
iv). Values are means ± SE; n = 5 rats/group.
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Effects of ET-1-receptor antagonists on HPV in blood- and
PSS-perfused rat lungs. There were no differences among the
respective control and experimental groups in the pressor responses to
5, 3, and 0% O2 before treatment of either blood- or
PSS-perfused lungs with either the vehicle (control), BQ-123, or BQ-788
(data not shown). In addition, baseline (normoxic) perfusion pressures were not affected by the ET-1-receptor antagonists in either blood- or
PSS-perfused lungs (data not shown). However, in blood-perfused lungs,
the response to hypoxia was inhibited by the ETA- but not by the ETB-receptor antagonist (Fig.
4). In contrast, in PSS-perfused lungs, the
response to hypoxia was inhibited equally by either BQ-123, BQ-788, or
the combination of both (Fig. 5). The
pressor response to exogenous ET-1 in the PSS-perfused lungs (17.2 ± 2.4 mmHg in control lungs) was unaffected by BQ-788 (17.8 ± 1.0 mmHg), partially reduced by BQ-123 (10.9 ± 2.0 mmHg), and eliminated by the combination of BQ-123 and BQ-788 (0.3 ± 0.1 mmHg; P < 0.05 vs. control value).

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Fig. 4.
BQ-123 but not BQ-788 inhibited hypoxic pulmonary vasoconstriction in
blood-perfused rat lungs. Successive hypoxic challenges with 10 min of
5, 3, and 0% O2 were given after addition of either
vehicle (n = 3 rats), 5 µM BQ-123 (n = 4 rats), or 5 µM BQ-788 (n = 4 rats) to perfusate. Pressure,
hypoxia-induced increase in lung perfusion pressure. Values are means ± SE. * Significant difference from respective control value,
P < 0.05 by ANOVA.
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Fig. 5.
BQ-123, BQ-788, and BQ-123+BQ-788 inhibited hypoxic pulmonary
vasoconstriction equally in physiological salt solution (PSS)-perfused
rat lungs. Successive hypoxic challenges with 10 min of 5, 3, and 0%
O2 were given after addition of either vehicle, 5 µM
BQ-123, 5 µM BQ-788, or BQ-123+BQ-788 to perfusate. Values are means ± SE; n = 4 rats/group. * Significant difference from
respective control value, P < 0.05 by ANOVA.
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In contrast to the preceding results, the responses to hypoxia were not
inhibited by the combined treatment with BQ-123 and BQ-788 in
PSS-perfused lungs costimulated with angiotensin II (Fig.
6). However, the pressor response to
angiotensin II was reduced slightly by the ET-1 antagonists (responses
to angiotensin II before and after treatment were 5.5 ± 0.8 and 5.1 ± 0.8 mmHg, respectively, in control lungs and 6.7 ± 1.3 and 4.0 ± 0.4 mmHg, respectively, in BQ-123+BQ-788-treated lungs; P < 0.05), and the response to exogenous ET-1 was blocked (response was
14.1 ± 1.8 mmHg in control lungs and 0.2 ± 0.1 mmHg in
BQ-123+BQ-788-treated lungs; P < 0.05).

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Fig. 6.
Costimulation with angiotensin II prevents inhibition of hypoxic
pulmonary vasoconstriction by BQ-123+BQ-788 in PSS-perfused rat lungs.
Successive hypoxic challenges with 10 min of 5, 3, and 0%
O2 were given after addition of either vehicle or 5 µM
BQ-123 plus 5 µM BQ-788 to perfusate. Lungs were stimulated with
bolus injection of 0.3 µg of angiotensin II 5 min before each hypoxic
challenge. Values are means ± SE; n = 4 rats/group.
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KATP-channel blocker prevents
inhibition of HPV by ETA-receptor
antagonist in both isolated lungs and intact rats. Glibenclamide did not affect normoxic perfusion pressure in either PSS- or
blood-perfused lungs (data not shown) but potentiated HPV in both
preparations [increases in HPV after either glibenclamide or
vehicle were 162 ± 32 and 50 ± 12%, respectively, in PSS-perfused
lungs (P < 0.05; n = 8 rats/group) and 95 ± 52 and 2 ± 8%, respectively, in blood-perfused lungs
(P < 0.05; n = 8 rats/group)]. Furthermore,
glibenclamide prevented the inhibition of HPV by BQ-123 in both PSS-
and blood-perfused lungs (Figs. 7 and
8, respectively).

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Fig. 7.
ATP-sensitive K+ (KATP)-channel blocker
glibenclamide (Glib) prevented inhibition of hypoxic pulmonary
vasoconstriction by BQ-123 in PSS-perfused rat lungs. Two successive
hypoxic challenges (10 min of 0% O2) were given before and
after addition of either Glib vehicle or 20 µM Glib and then either
BQ-123 vehicle or 5 µM BQ-123 to perfusate. The 2 hypoxic responses
before and after each treatment in each lung were averaged. Values are
means ± SE; n = 4 rats/group. * Significant difference
from respective vehicle value, P < 0.05 by ANOVA.
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Fig. 8.
Glib prevented inhibition of hypoxic pulmonary vasoconstriction by
BQ-123 in blood-perfused rat lungs. Two successive hypoxic challenges
(10 min of 0% O2) were given before and after addition of
either Glib vehicle or 50 µM Glib and then either BQ-123 vehicle or 5 µM BQ-123 to perfusate. The 2 hypoxic responses before and after each
treatment in each lung were averaged. Values are means ± SE;
n = 4 rats/group. * Significant difference from respective
vehicle value, P < 0.05 by ANOVA.
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The effects of glibenclamide on baseline (normoxic) systemic and
pulmonary hemodynamics and the responses to acute hypoxia in
catheterized rats are shown in Table 4.
Glibenclamide increased systemic arterial pressure and total systemic
and pulmonary resistances and decreased heart rate but had no effect on
HPV. Subsequent treatment with BQ-123 decreased systemic and pulmonary
arterial pressures in both vehicle- and glibenclamide-treated rats
(Table 5). Although the
ETA-receptor antagonist inhibited HPV in control rats, it
failed to do so in the glibenclamide-treated animals (Table 5, Fig.
9).

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Fig. 9.
Glib prevented inhibition of hypoxic pulmonary vasoconstriction by
BQ-123 in rats. Hypoxic challenges (5-6 min of 10%
O2) were given before and 45 min after treatment with
either BQ-123 vehicle or BQ-123 (50 µg · kg 1 · min 1
iv) in rats pretreated with either Glib vehicle or Glib (20 mg/kg iv).
Values are means ± SE; n = 6 rats/group. * Significant
difference from respective before value, P < 0.05 by ANOVA.
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DISCUSSION |
The major findings of this study were that HPV was inhibited by an
ETA-receptor antagonist in both intact rats and isolated rat lungs and that the inhibition of HPV by ETA-receptor
blockade was prevented by pretreatment with the
KATP-channel blocker glibenclamide. These results suggest
that ET-1 is involved in the mechanism of HPV both in vivo and in vitro
through an ETA receptor-mediated suppression of
KATP-channel activity.
It has been previously reported (4, 5, 9, 30, 41, 46) that either
selective ETA- or nonselective ETA- and
ETB-receptor antagonists but not selective
ETB-receptor antagonists inhibit HPV in intact animals
including rats. In contrast, ETA-receptor antagonists were
found not to inhibit HPV in PSS-perfused rat lungs (17, 40). Because
this disparity raises the possibility that the mechanism of HPV differs
between the in vivo and in vitro preparations, the first aim of our
study was to reassess the effects of ET-1 antagonists on HPV in both
intact rats and isolated rat lungs. Our results in rats confirmed that
either a selective ETA- or a nonselective ETA-
or ETB-receptor antagonist but not a selective ETB-receptor antagonist markedly attenuated HPV. Our
isolated lung studies also showed that HPV was significantly reduced by only an ETA-receptor antagonist in blood-perfused lungs and
by either an ETA- or an ETB-receptor antagonist
in PSS-perfused lungs. These results indicate that
ETA-receptor activation by endogenous ET-1 is involved in
the mechanism of HPV in both intact rats and isolated rat lungs.
On the basis of evidence that activation of KATP channels
inhibits HPV (3, 7, 11, 29, 45) and that ET-1 can reduce KATP-channel activity through stimulation of
ETA receptors (18, 26, 43), the second aim of our study was
to test whether the inhibition of HPV by ETA-receptor
blockade could be attributed to increased KATP-channel
activity. The results showed that the KATP-channel blocker
glibenclamide prevented inhibition of HPV by the
ETA-receptor antagonist BQ-123 in intact rats and in both blood- and PSS-perfused lungs. Because glibenclamide did not potentiate HPV in rats and caused only moderate augmentation in perfused lungs,
its prevention of the BQ-123-induced inhibition was not due simply to a
more vigorous hypoxic response. Instead, we believe that the findings
support the idea that ETA-receptor blockade reverses an
endogenous ET-1-mediated suppression of vascular smooth muscle
KATP-channel activity that, in turn, leads to increased K+ current and inhibition of hypoxia-induced membrane
depolarization. Whether the evident ET-1-mediated suppression of
KATP-channel activity occurs basally, i.e., during
normoxia, or is associated with a hypoxia-induced release of ET-1 is
unclear from our results. However, previous studies (12, 38) in
isolated rat lungs and intact rats indicated that the levels and
duration of hypoxia used in our experiments caused little or no
increase in either plasma or lung tissue levels of ET-1. In addition,
glibenclamide did not elicit a hypoxia-like pulmonary vasoconstriction
during normoxia in either lungs or rats (the increased total pulmonary resistance after glibenclamide in rats was associated with a decrease in cardiac output rather than with an increase in pulmonary arterial pressure). This suggests that HPV is not due simply to inhibition of
KATP channels. Thus our speculation is that a basal level
of ET-1-mediated suppression of KATP-channel activity
"allows" HPV rather than that a hypoxia-induced release of ET-1
inhibits KATP channels and "causes" HPV (1).
A major difference between our isolated rat lung protocol and that of
previous investigators who observed no inhibition of HPV by the
ETA-receptor antagonists BQ-123 (40) and FR-139317 (17) was
that in the earlier studies the PSS-perfused lungs were stimulated with
angiotensin II both before and after addition of the blockers. We
performed a similar experiment and found that although stimulation with
angiotensin II eliminated the blunting of HPV by the combination of
BQ-123 and BQ-788, it did not interfere with the inhibition of the
vasoconstrictor response to exogenous ET-1; i.e., it did not prevent
ET-1-receptor blockade. Angiotensin II has often been used to enhance
the hypoxic pressor responsiveness of PSS-perfused rat lungs, and our
finding indicates that it modifies reactivity of the preparation so as
to render the hypoxic response insensitive to inhibition by
ET-1-receptor blockade. Among its many actions, angiotensin II also
inhibits vascular smooth muscle K+ channels, including
KATP channels (6, 19, 24, 25), and it is possible that it
prevents the inhibition of HPV at least partly by suppressing
K+-channel activation and membrane hyperpolarization after
ET-1-receptor blockade. In other words, adding exogenous angiotensin II
to PSS-perfused lungs appears to circumvent the role of endogenous
ET-1.
The ETA-receptor antagonist BQ-123 had similar inhibitory
effects on HPV in all three preparations, i.e., in catheterized rats
and both blood- and PSS-perfused rat lungs, but there was a
preparation-dependent difference in the effect of the
ETB-receptor antagonist BQ-788. Whereas BQ-788 did not
inhibit HPV in either rats or blood-perfused lungs, it was as effective
as BQ-123 in reducing HPV in PSS-perfused lungs. Because the equivalent
inhibition of HPV by BQ-123 and BQ-788 in PSS-perfused lungs differed
from the respective effect of the antagonists on the vasoconstrictor response to exogenous ET-1, i.e., the response to ET-1 was reduced slightly by BQ-123 but unaffected by BQ-788, BQ-788 was apparently not
mimicking the effect of BQ-123 by blocking ETA receptors. Whether inhibition of HPV by ETB-receptor blockade was also
due to activation of KATP channels or to some other effect
cannot be determined from our study. There is evidence for
ETB receptor-mediated inhibition of both Ca2+-
and voltage-sensitive K+ currents in isolated rat pulmonary
arterial smooth muscle cells (21, 35, 37), and perhaps
ETB-receptor blockade inhibited HPV in PSS-perfused lungs
by increasing the activity of these channels. We have no explanation
for why this might have occurred in PSS- but not in blood-perfused
lungs. In this regard, it has been previously noted that the nature of
the rat lung perfusate, i.e., blood versus PSS, determines whether
exogenous ET-1 constricts predominantly pre- or postcapillary vessels
(28, 34).
BQ-123 inhibited HPV in both rats and isolated lungs, but the magnitude
of inhibition appeared to be greater in rats than in lungs (for
example, compare the difference in HPV before and after BQ-123 in rats
in Fig. 9 with that in PSS- and blood-perfused lungs in Figs. 7 and 8,
respectively). We do not have a definitive explanation for this
difference, but one possibility is that the circulating level of an
endogenous activator of KATP channels, such as calcitonin
gene-related peptide, adenosine, or catecholamines (39), was higher in
rats than in isolated lungs. Prostacyclin, another activator of
KATP channels (39), is not a likely candidate because there
was no apparent difference in the inhibition of HPV by BQ-123 between
PSS-perfused lungs that were treated with the cyclooxygenase inhibitor
meclofenamate to enhance hypoxic pressor responsiveness and
blood-perfused lungs that were not treated.
Our interpretation that endogenous ET-1 promotes HPV by suppressing
vascular smooth muscle KATP-channel activity needs to be
reconciled with earlier evidence (11) in rat lungs that relatively low
doses of exogenous (intraluminal) ET-1 cause at least transient inhibition of HPV by stimulating KATP channels. Although
the relative roles of the endothelium-derived vasodilators nitric
oxide, prostacyclin, and hyperpolarizing factor remain unclear,
subsequent studies in rat lungs and pulmonary arteries have shown that
the transient ET-1-induced pulmonary vasodilation is due to
ETB-receptor-mediated activation of endothelial cells (8,
14, 20, 27, 33, 36). Thus a possible explanation of the apparent
paradox is that exogenous ET-1 causes a transient ETB
receptor-mediated increase in release of an endothelium-derived
vasodilator that counteracts the endogenous ET-1-induced,
ETA receptor-mediated suppression of KATP activity.
In summary, this study showed that ETA-receptor blockade
inhibited HPV in both intact rats and isolated rat lungs and that the
inhibition of HPV was, in turn, prevented by inhibition of KATP channels. These results indicate that endogenous
ET-1-induced activation of ETA receptors is a component of
the mechanism of HPV. Because ETA-receptor blockade
inhibited HPV without blocking ET-1-induced vasoconstriction and
because the pulmonary vasoconstrictor response to ET-1 is long lasting,
whereas that to hypoxia is rapidly reversed (11, 34), we reason that
instead of mediating HPV in the classic sense, endogenous ET-1 promotes
HPV by suppressing vascular smooth muscle KATP-channel
activity and thereby allowing a direct hypoxia-induced inhibition of a
voltage-sensitive K+ channel to cause membrane
depolarization and activation of L-type Ca2+ channels (2,
31, 32, 47). This interpretation reconciles the apparently disparate
observations that although hypoxia can act directly on isolated
peripheral pulmonary arterial smooth muscle cells to cause membrane
depolarization and Ca2+ influx, HPV is inhibited by
ETA-receptor blockade in intact animals and isolated lungs.
 |
ACKNOWLEDGEMENTS |
This study was supported by National Heart, Lung, and Blood
Institute Grants HL-14985 (to I. F. McMurtry) and HL-48038 (to D. M. Rodman) and Parke-Davis Pharmaceutical Research (I. F. McMurtry).
 |
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: I. F. McMurtry,
CVP Research Laboratory, B-133, Univ. of Colorado Health Science
Center, 4200 East Ninth Ave., Denver, CO 80262 (E-mail:
Ivan.McMurtry{at}uchsc.edu).
Received 26 July 1999; accepted in final form 9 September 1999.
 |
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