Vol. 276, Issue 2, H383-H390, February 1999
Endothelium-independent vascular relaxation mediating
ETB receptor in rabbit mesenteric
arteries
Takanori
Iwasaki,
Mitsuru
Notoya,
Yoko
Hayasaki-Kajiwara,
Toshitake
Shimamura,
Noriyuki
Naya,
Mitsuyoshi
Ninomiya, and
Masatoshi
Nakajima
Discovery Research Laboratories II, Shionogi & Company Limited,
Osaka 561-0825, Japan
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ABSTRACT |
Vascular response
mediating endothelin (ET)B
receptor was studied using isolated rabbit mesenteric arteries. ET-1
(0.1-30 nM) caused a concentration-dependent contraction, whereas
ET-3 >100 nM caused only weak contraction. Up to 1 µM of
sarafotoxin S6c showed no contraction. In arteries precontracted with
phenylephrine, ET-3 (0.03-1 nM) caused a concentration-dependent
relaxation, which was not affected by endothelium denudation. The
ET-3-induced relaxation was antagonized by BQ-788 and PD-142893 but not
by BQ-123 in the endothelium-denuded arteries. Treatment with
indomethacin but not with
NG-nitro-L-arginine methyl ester
completely inhibited the relaxation. ET-3 stimulated the release of
6-keto-PGF1
and
PGE2 from the endothelium-denuded
arteries. ET-3 also significantly increased cAMP content but not cGMP
content in the arteries. Radioligand-binding studies using serial
sections of the artery revealed the expression of not only
ETA but also
ETB receptors in the smooth muscle
layer of the arteries. These results suggest that ET-3 activates
ETB receptor in smooth muscle
cells of rabbit mesenteric artery, producing vasodilator prostaglandins
from arachidonic acid probably via a catalysis of cyclooxygenase, which
accumulates cAMP in subendothelial tissues and produces relaxations.
endothelin; prostaglandin I2; adenosine 3',5'-cyclic monophosphate
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INTRODUCTION |
ENDOTHELIN (ET) was originally discovered as a most
potent vasoconstrictor peptide released from vascular endothelial cells (36). The ET family is composed of 21-residue peptide isoforms of ET-1,
ET-2, and ET-3 (10). The responsiveness to ET isopeptides is
heterogeneous in a variety of vascular and nonvascular tissues. The
physiological and pathophysiological actions of ETs are mediated by at
least two distinct receptor subtypes,
ETA and
ETB receptors, both of which have
been cloned (1, 16, 24). The ETA
receptor has a higher affinity for ET-1 and ET-2 than for ET-3, whereas the ETB receptor has an equal
affinity for all isopeptides (1, 24). Furthermore, the receptor for
ET-3 has been cloned from Xenopus
laevia dermal melanophores (13), but its physiological role remains unknown. The ETA
receptor located in vascular smooth muscle mediates vasoconstrictions
induced by ET-1 (15). Activation of the
ETB receptor located in vascular
smooth muscle also induces constriction in various vessels, such as the
rabbit jugular vein (29), rat renal artery (22), rabbit saphenous vein
(7, 28), rabbit pulmonary artery (33), and human saphenous vein (20).
ETA and
ETB receptors that contribute to
the contractile responses vary greatly in their vasculature
distribution. On the other hand, the
ETB receptor, which is located in
the endothelium and releases endothelium-derived relaxing substances
such as nitric oxide and PGI2, is
also considered to mediate the vasorelaxation induced by ET-1 or ET-3
(6, 34). Recently, these functional heterogeneous
ETB receptors present in the
vascular endothelium and smooth muscle have been pharmacologically
classified into ETB1 and ETB2 subtypes,
respectively (28, 33).
Endothelium-independent vascular relaxation mediated by
ETB receptor has not yet been
reported. In this study, we offer the first evidence that the
endothelium-independent vascular relaxation induced by ET-3 is mediated
by the ETB receptor in rabbit
mesenteric arteries. We also present a description of the mechanism of
the relaxation and the expression of not only the
ETA receptor but also the
ETB receptor in the smooth muscle
layer of the arteries using a quantitative autoradiographic technique.
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MATERIALS AND METHODS |
Arterial ring preparation. Male Japan
White rabbits (Kitayama Labes Breeding Laboratories, Minowa, Japan),
weighing 2.8-3.8 kg, were anesthetized by intravenous injections
of pentobarbital sodium (35 mg/kg) and killed by exsanguination from
common carotid arteries. Superior mesenteric arteries, distal femoral
arteries, pulmonary arteries, and thoracic aortas were isolated.
Intrarenal, interlobar branches of the renal arteries were also
isolated from the kidney. The arteries were cleaned of the surrounding
tissue and cut into rings ~3 mm in length. One to four pairs of rings were prepared with and without endothelium. Removal of the endothelium was performed by gently rubbing the intimal surface with a stainless steel wire. The ring was mounted in a muscle bath containing
Krebs-Henseleit solution, which was maintained at 37 ± 0.3°C
and aerated with a mixture of 95%
O2-5%
CO2. The hook anchoring the upper
end of the rings was connected to the lever of a force-displacement
transducer (TB-611T, Nihon Kohden, Tokyo, Japan). Resting tension was
adjusted to 2.0 g. Constituents of the solution were as follows (in
mM): 118 NaCl, 4.7 KCl, 1.2 KH2PO4,
1.2 MgSO4, 2.5 CaCl2, 25.0 NaHCO3, and 11.0 glucose. The pH
of the solution was 7.35-7.42. Before the start of each
experiment, the preparation was allowed to equilibrate in the bathing
media for 60-90 min, during which time the solution was replaced
every 10-15 min.
Isometric contractions and relaxations were recorded on a polygraph
recorder (WT-685G, Nihon Kohden). The contractile response to 50 mM
K+ was first obtained, and the
preparations were repeatedly washed with fresh media and equilibrated.
The concentration-response curves for ET-1, ET-3, sarafotoxin S6c,
IRL-1620, PGI2,
PGE2, and acetylcholine were
obtained by adding the compounds directly to the bathing media in
cumulative concentrations. To test the relaxant response, the
preparations were partially contracted with phenylephrine; the
contraction was between 25 and 50% of that induced by 50 mM
K+. After the end of each
experiment on the relaxant effect of agonist, 100 µM papaverine was
added to attain the maximal relaxation. The papaverine-induced
relaxation and the K+ (50 mM)-induced contraction were taken as 100% for relaxant and contractile responses to the test drugs, respectively. Preparations were treated for 30 min with blocking agents before the
concentration-response curves for agonists were obtained. The responses
of endothelium-denuded rings were compared with those of rings with
intact endothelium obtained from the same rabbits. Removal of the
endothelium was confirmed by the disappearance of relaxation induced by
acetylcholine (100 nM). In seven endothelium-denuded mesenteric
arteries, the absence of endothelial cell demarcation was confirmed
histologically by a silver staining procedure (3). The concentration of
an agonist causing EC50 was
calculated from each concentration-response curve using Probit analysis
(9).
Measurements of
6-keto-PGF1 and
PGE2.
The endothelium-denuded preparations were equilibrated in 1 ml of
Krebs-Henseleit solution aerated with a mixture of 95%
O2-5% CO2 at 37°C for 120 min before
the start of the experiment. The incubation solution was replaced every
20-25 min. After preincubation for 30 min, the preparation was
incubated with ET-3 (1-10 nM) for 30 min. The concentration of
6-keto-PGF1 or
PGE2 in the incubation solution
was measured using a commercial enzyme-linked immunosorbent assay
(ELISA) kit (Amersham, Buckinghamshire, UK).
Determination of cyclic nucleotide
contents. After the absence of endothelium had been
confirmed by the absence of a relaxant response to acetylcholine (100 nM), the relaxant response of ET-3 (3 nM) was obtained. Just after the
relaxation had reached maximum, the artery mounted on the hook was
instantly frozen with a punch chilled with liquid nitrogen. The tissues
were homogenized in ice-cold 6% trichloroacetic acid (TCA) with a
Polytron homogenizer. After centrifugation at 1,700 g for 15 min at 4°C, an ether
extraction procedure was carried out three times on the supernatant.
The extract was then frozen, dried, and then dissolved in 50 mM acetate buffer (pH 5.8). An aliquot of the dissolved solution was used to
determine the amounts of cAMP and cGMP using a commercial ELISA kit
(Amersham). After the TCA-precipitated pellets were solubilized with
0.5 M sodium hydroxide, the protein concentration was measured by the
Bradford method (2).
Quantitative in vitro autoradiography.
The isolated superior mesenteric arteries were frozen in dry-ice
powder. Serial frozen sections (20 µm) were cut on a cryostat at
20°C and thaw mounted onto
poly-L-lysine-coated slides.
After preincubation for 15 min in the incubation solution containing 20 mM HEPES (pH 7.4), 135 mM NaCl, 2 mM
CaCl2, and 0.2% bovine serum
albumin and 0.01% bacitracin, the sections were incubated with the
solution containing 125I-labeled
ET-1 (10-100 pM) in the absence and presence of BQ-123 (1 µM) or
125I-labeled IRL-1620 (10-120
pM) for 160 min at room temperature. Nonspecific binding was determined
using serial sections in the presence of an excess concentration (0.3 µM) of either unlabeled ET-1 or IRL-1620. After incubation, the
sections were washed four times in ice-cold buffer, followed by a dip
in ice-cold distilled water, and then rapidly dried under a stream of
cold air. The dried sections were placed in X-ray cassettes and exposed
to X-ray films (RX, Fuji Photo Film, Tokyo, Japan) with calibrated
125I-labeled standards as shown
previously (37). After the film was developed, the optical density of
the smooth muscle layer in the autoradiograms was quantified using an
Optimas computer-assisted image analysis system (Optimas, Bothell, WA).
The bound radioactivity was calculated with standard curves. The
results were expressed as attomoles per millimeter squared.
Drugs and reagents. Drugs used were
BQ-123 and BQ-788 (Neosystem, Strasbourg, France); ET-1, ET-3,
sarafotoxin S6c, IRL-1620, and PD-142893 (Peptide Institute, Osaka,
Japan); adenosine, aspirin, NG-nitro-L-arginine
(L-NNA),
NG-nitro-L-arginine methyl ester
(L-NAME), bovine hemoglobin,
phenylephrine hydrochloride, PGE2,
and PGI2 sodium salt (Sigma, St.
Louis, MO); indomethacin, sodium nitroprusside, and TCA (Nacalai
Tesque, Osaka, Japan); acetylcholine chloride (Dainippon
Pharmaceutical, Osaka, Japan); pentobarbital sodium (Abbott, North
Chicago, IL); and 125I-labeled
ET-1 ([125I]ET-1,
2,200 Ci/mmol) and 125I-labeled
IRL-1620
([125I]IRL-1620, 2,200 Ci/mmol) (New England Nuclear, Boston, MA). Oxyhemoglobin (OxyHb) was
prepared from bovine hemoglobin according to the method described by
Martin et al. (14).
Statistical analyses. The results
shown in the text and Figs. 1-5 were expressed as means ± SE.
Statistical analyses were done using the Tukey's method after one-way
analysis of variance (32).
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RESULTS |
Responses to ET-1, ET-3, and sarafotoxin
S6c. The addition of ET-1 in concentrations ranging
from 0.1 to 30 nM produced a concentration-dependent contraction in
endothelium-intact arteries. Further increases of the concentration of
ET-1 did not show any contractions (Fig.
1). Endothelium denudation did not affect
the response; the mean values of the maximum contraction and
EC50 in endothelium-intact
arteries were 75.7 ± 5.9% (n = 5)
and 1.2 ± 0.2 nM (n = 5),
respectively, and those in endothelium-denuded arteries were 70.7 ± 8.2% (n = 5) and 1.1 ± 0.3 nM
(n = 5), respectively. On the other
hand, the addition of ET-3 above 100 nM caused only weak contractions
in both arteries. Contractions induced by 1 µM ET-3 in
endothelium-intact and -denuded arteries were 12.1 ± 8.4%
(n = 5) and 6.0 ± 1.9%
(n = 5), respectively. In contrast, up
to 1 µM of sarafotoxin S6c (an
ETB receptor agonist) had no effect in either type of artery.
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Fig. 1.
Concentration-contractile response curves for endothelin-1 (ET-1,
circles), ET-3 (triangles), and sarafotoxin S6c (squares) in
endothelium-intact (open symbols) and -denuded (closed symbols) rabbit
mesenteric arteries. Responses to ET-1, ET-3, and sarafotoxin S6c were
obtained under resting conditions. Contractions induced by 50 mM
K+ were taken as 100%; mean
absolute values in endothelium-intact and -denuded arteries in response
to ET-1 were 4.3 ± 0.2 g (n = 5)
and 4.7 ± 0.3 g (n = 5),
respectively, those in response to ET-3 were 4.4 ± 0.2 g
(n = 5) and 4.7 ± 0.2 g
(n = 5), respectively, and those in
response to sarafotoxin S6c were 4.4 ± 0.2 g
(n = 5) and 4.8 ± 0.4 g
(n = 5), respectively.
Vertical bars are means ± SE.
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Figure 2 shows the typical recordings of
the responses to acetylcholine and ET-3 in endothelium-intact and
-denuded arteries. In the endothelium-intact arteries partially
contracted with phenylephrine, acetylcholine ranging from 1 to 100 nM
caused a concentration-dependent relaxation (Fig.
2A,
left). In the same preparation, ET-3
(0.03-1 nM) elicited a concentration-dependent relaxation, whereas
ET-3 >3 nM reversed to contraction (Fig.
2A,
right). Acetylcholine-induced relaxations were reversed to concentration-dependent contractions by
removal of the endothelium (Fig. 2B,
left). However, the ET-3-induced relaxation was not affected by removal of the endothelium (Fig. 2B,
right). Quantitative data of these
responses to acetylcholine and ET-3 are summarized in Fig.
3. The maximum relaxation was induced by
100 nM acetylcholine in the endothelium-intact arteries (92.0 ± 1.5%, n = 10), whereas the same
concentration of acetylcholine caused contraction in the
endothelium-denuded arteries. The concentration-dependent response
curves of ET-3 in endothelium-intact and -denuded arteries did not
differ significantly; the mean values of the maximum relaxations induced by ET-3 and the mean values of
EC50 for the relaxant response in
endothelium-intact arteries were 62.8 ± 4.9%
(n = 10) and 0.16 ± 0.04 nM
(n = 10), respectively, and those
values in endothelium-denuded arteries were 75.2 ± 3.6%
(n = 10) and 0.17 ± 0.04 nM
(n = 10), respectively. ET-1
(0.03-1 nM) also caused concentration-dependent relaxation in both
endothelium-intact and -denuded arteries; the mean values of the
maximum relaxation induced by 1 nM ET-1 and the mean values of
EC50 for the relaxant response in
endothelium-intact arteries were 87.3 ± 6.3%
(n = 3) and 0.08 ± 0.02 nM
(n = 3), respectively, and those
values in endothelium-denuded arteries were 85.2 ± 6.6%
(n = 3) and 0.07 ± 0.02 nM
(n = 3), respectively. Furthermore,
sarafotoxin S6c (0.03-1 nM) and IRL-1620 (0.3-10 nM),
selective ETB receptor agonists,
also induced relaxations in endothelim-denuded arteries; the mean
values of the maximum relaxation induced by 1 nM sarafotoxin S6c and
the mean values of EC50 for the
relaxant response were 54.9 ± 7.6%
(n = 5) and 0.26 ± 0.08 nM
(n = 5), respectively, and those
induced by 10 nM IRL-1620 and those of
EC50 were 46.3 ± 4.9%
(n = 6) and 2.44 ± 0.31 nM (n = 6), respectively.
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Fig. 2.
Responses to ACh and endothelin (ET)-3 in endothelium-intact and
-denuded mesenteric arteries obtained from same rabbit. Responses to
ACh and ET-3 were obtained using arteries partially contracted with
phenylephrine. Traces are responses to the following:
A)
left, ACh;
right, ET-3 in endothelium-intact
arteries; B)
left, ACh;
right, ET-3 in endothelium-denuded
arteries; C)
left, ACh;
right, ET-3 in endothelium-denuded
arteries treated with 3 µM BQ-123;
D)
left, ACh;
right, ET-3 in endothelium-denuded
arteries treated with 3 µM BQ-788. Dotted lines under each tracing
represent the level after addition of 100 µM papaverine (PA).
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Fig. 3.
Concentration-response curves for ACh
(A) and ET-3
(B) in endothelium-intact ( ) and
-denuded ( ) rabbit mesenteric arteries. Responses to ACh and ET-3
were obtained in arteries partially contracted with phenylephrine.
Contractions induced by 50 mM K+
were taken as 100% contraction; mean absolute value in
endothelium-denuded arteries in response to acetylcholine was 4.1 ± 0.2 g (n = 10). Relaxations caused by
100 µM papaverine were taken as 100% relaxation; mean absolute value
in endothelium-intact arteries in response to acetylcholine was 1.0 ± 0.1 g (n = 10), and those in
endothelium-intact and -denuded arteries in response to ET-3 were 1.6 ± 0.2 g (n = 10) and 1.7 ± 0.2 g (n = 10), respectively. Vertical
bars are means ± SE.
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The endothelium-independent vascular relaxation induced by ET-3 was not
observed in thoracic aortas (n = 3), pulmonary arteries (n = 3),
renal arteries (n = 3), and
femoral arteries (n = 3).
Effects of ET antagonists L-NAME
and indomethacin on relaxant response to ET-3.
As shown in Fig. 2C, relaxation
induced by ET-3 in endothelium-denuded arteries was not inhibited by
treatment with 3 µM BQ-123, a selective
ETA receptor antagonist, whereas
treatment with 3 µM BQ-788, a selective
ETB receptor antagonist,
completely abolished the relaxation (Fig.
2D). Quantitative data are
summarized in Fig.
4A. The
concentration-response curves of ET-3 shifted to the right by treatment
with 3 µM PD-142893, an
ETA/ETB
receptor antagonist. Furthermore, treatment with 1 µM indomethacin, a
cyclooxygenase inhibitor, completely abolished the ET-3-induced
relaxation and reversed it to a weak contraction, whereas the
relaxation was not inhibited by treatment with 100 µM
L-NAME, a nitric oxide synthase
inhibitor (Fig. 4B). The effects of
these ET receptor antagonists on ET-3-induced relaxation in
endothelium-intact arteries were similar to those in
endothelium-denuded arteries (data not shown). Treatment with
L-NAME (100 µM) did not affect
the ET-3-induced relaxation even in endothelium-intact arteries; the
mean values of the maximum relaxation induced by 1 nM ET-3 and the mean
values of EC50 for the relaxant
response in the absence of
L-NAME were 67.3 ± 8.6%
(n = 6) and 0.10 ± 0.01 nM
(n = 6), respectively, and those in
the presence of L-NAME were 61.1 ± 5.3% (n = 6) and 0.15 ± 0.04 nM (n = 6), respectively. The
ET-3-induced relaxation in the arteries was also not inhibited by
treatment with L-NNA (10 µM),
another nitric oxide synthase inhibitor (31); the mean values of the
maximum relaxation induced by 1 nM ET-3 and the mean values of
EC50 for the relaxant response in
the absence of L-NNA were 62.7 ± 8.5% (n = 7) and 0.16 ± 0.05 nM (n = 7), respectively, and those in the presence of
L-NNA were 53.4 ± 9.0%
(n = 7) and 0.29 ± 0.09 nM
(n = 7), respectively. Furthermore,
treatment with 10 µM OxyHb (14) did not affect the ET-3-induced
relaxation (n = 7). In
endothelium-intact arteries, the ET-3-induced relaxation was also
completely inhibited by indomethacin
(n = 6). Treatment with 1 µM
indomethacin did not attenuate the relaxations induced by adenosine
(0.01-1 µM, n = 4),
PGI2 (0.03-10 nM,
n = 4) and sodium nitroprusside
(1-100 nM, n = 4) in the
arteries. Abolishment of the ET-3-induced relaxation was also observed
in the arteries treated with 50 µM aspirin
(n = 7), another cyclooxygenase
inhibitor (30).
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Fig. 4.
Modifications by BQ-123, BQ-788, and PD-142893
(A) and
NG-nitro-L-arginine methyl ester
(L-NAME) and indomethacin
(B) of relaxant response to ET-3 in
endothelium-denuded rabbit mesenteric arteries.
A: control (), BQ-123 (3 µM,
), BQ-788 (3 µM,  ), and PD-142893 (3 µM,  );
B: control (),
L-NAME (100 µM, ), and
indomethacin (1 µM, ) were applied 30 min before application of
ET-3. Response to ET-3 was obtained in arteries partially contracted
with phenylephrine. Relaxations induced by 100 µM papaverine were
taken as 100% relaxation; mean absolute values in control arteries and
those treated with BQ-123, BQ-788, and PD-142893 were 1.8 ± 0.2 g
(n = 8), 1.7 ± 0.1 g
(n = 6), 1.9 ± 0.2 g
(n = 6), and 2.2 ± 0.2 g
(n = 5), respectively, and in control
arteries and those treated with
L-NAME and indomethacin were 1.6 ± 0.2 g (n = 5), 1.7 ± 0.2 g
(n = 5), and 1.7 ± 0.4 g
(n = 5), respectively.
Contractions induced by 50 mM K+
were taken as 100% contraction; mean absolute values in arteries
treated with BQ-788 and indomethacin were 3.2 ± 0.4 g
(n = 5) and 4.0 ± 0.3 g
(n = 5), respectively. Vertical bars
are means ± SE.
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In endothelium-denuded mesenteric arteries,
PGI2 (0.03-10 nM) and
PGE2 (0.003-1 nM) induced
concentration-dependent relaxations; the mean values of the relaxation
induced by 10 nM PGI2 and the mean
values of EC50 for relaxant
responses were 89.6 ± 1.0% (n = 5) and 1.4 ± 0.4 nM (n = 5),
respectively, and those induced by 1 nM
PGE2 and those of
EC50 were 84.3 ± 2.6%
(n = 5) and 58.8 ± 12.4 pM
(n = 5), respectively.
Effects of ET-3 on prostaglandin release and
nucleotide contents. The addition of ET-3 in
concentrations ranging from 1 to 10 nM increased the release of
6-keto-PGF1 and
PGE2 from endothelium-denuded
arteries in a concentration-dependent manner. The increments of the
6-keto-PGF1 and
PGE2 stimulated by 10 nM ET-3 were
4.4- and 1.9-fold compared with those of the control, respectively
(Table 1). As shown in Table
2, 3 nM ET-3 significantly increased the cAMP content
approximately fourfold compared with that of the control, whereas the
cGMP content was not affected by ET-3. Indomethacin (1 µM) had no
effect on these basal nucleotide contents. Treatment with indomethacin
abolished the increment of the cAMP content caused by 3 nM ET-3,
whereas the cGMP content was slightly increased by combined treatment
with indomethacin and ET-3.
Quantitative in vitro autoradiographical
study. Specific
[125I]ET-1 binding
sites were observed in the media and in the perivascular structures of
the artery, whereas the binding to the intimal layer was not
detectable. The specific binding of
[125I]ET-1 in the
presence of 1 µM BQ-123 and that of
[125I]IRL-1620,
a specific ETB receptor agonist,
were also detected in these layers. Saturation binding curves of
[125I]ET-1 in the
absence and presence of BQ-123 (1 µM) and the curves of
[125I]IRL-1620 in
smooth muscle layer are shown in Fig. 5,
A and
B. Scatchard analyses of
[125I]ET-1 binding
showed that the apparent dissociation constant (Kd) and
maximal binding (Bmax) values in
the absence of BQ-123 were 39.1 ± 5.7 pM
(n = 4) and 169.0 ± 10.6 amol/mm2 (n = 4), respectively (Fig.
5A, inset), and those values in the presence of
BQ-123 were 32.4 ± 5.4 pM (n = 3)
and 17.1 ± 1.6 amol/mm2
(n = 3), respectively (Fig.
5B,
inset). The apparent
Kd and
Bmax values obtained from the
Scatchard analysis of
[125I]IRL-1620 binding
were 46.9 ± 4.5 pM (n = 4) and
14.3 ± 1.7 amol/mm2
(n = 4), respectively (Fig.
5B,
inset). The
Bmax value of
[125I]ET-1 in the
presence of BQ-123 correlated well with that of [125I]IRL-1620
binding. Combined treatment with BQ-788 (1 µM) and BQ-123 (1 µM)
completely displaced the specific binding of
[125I]ET-1 to the
sections. Thus the calculated ratio of the
ETA to ETB receptor number on the artery
was ~10:1.
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Fig. 5.
Saturation binding curves of
[125I]ET-1
(A) and
[125I]ET-1 in presence
of BQ-123 and
[125I]IRL-1620
(B) in consecutive sections of
rabbit mesenteric artery. Increasing concentrations of
[125I]ET-1 were
applied to sections (20 µm) in absence
(A, ) and presence
(B, ) of 1 µM BQ-123. Increasing
concentrations of
[125I]IRL-1620 ( )
were applied to sections (20 µm). Scatchard plot for specific binding
of [125I]ET-1 is shown
in inset of
A, and those for specific binding of
[125I]ET-1 in presence
of BQ-123 or
[125I]IRL-1620 in
inset of
B.
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DISCUSSION |
Vasocontractile and vasorelaxant responses to ET isopeptides have
already been reported to be mediated mainly by the
ETA receptor located in the smooth
muscle and by the ETB receptor
located in the endothelium, respectively (16). Furthermore, the
vasoconstriction via ETA receptor
activation by ET-1 is known to be modulated by endothelium-derived
relaxing substances, such as nitric oxide and
PGI2, released basally and through
the activation of endothelial ETB
receptor (26, 38). In the present study, ET-1 induced a
concentration-dependent contraction in rabbit mesenteric arteries, which was not affected by removal of the endothelium. Furthermore, ET-3
showed a weak contraction, whereas sarafotoxin S6c, a selective ETB receptor agonist, showed none.
The ETA receptor has been shown to
display a greater selectivity for ET-1 and ET-2 than for ET-3, whereas
the ETB receptor shows almost
equal affinity for all three isopeptides (1, 24). From the agonist
selectivity for each receptor subtype, our findings suggest that the
ET-1-induced contraction of rabbit mesenteric artery is mainly mediated
by the ETA receptor, and the
endothelium does not play a significant role in counteracting the
contraction through the release of relaxing substances.
Vasorelaxations induced by ET isopeptides have been reported in various
vessels such as rat thoracic aorta (12, 18), rat mesenteric artery
(34), rat basilar artery (25), and rabbit lateral saphenous vein (7).
The relaxation induced by ET-3 in these vascular preparations is
thought to be mediated by the release of vasodilating substance via
activation of the ETB receptor located in the endothelium. In this study, we showed that ET-3 caused a
concentration-dependent relaxation in rabbit mesenteric arteries
precontracted with phenylephrine, and the relaxation was not influenced
by the removal of the endothelium (Fig.
3B). ET-1, sarafotoxin S6c, and
IRL-1620 also induced the endothelium-independent relaxation in the
arteries. Such relaxation induced by ET-3 was abolished by BQ-788
treatment, a selective ETB
receptor antagonist, and was attenuated by PD-142893 treatment, an
ETA/ETB
receptor antagonist. However, no effect was observed with a high
concentration of BQ-123, a selective
ETA receptor antagonist (Fig.
4A). These results strongly suggest
that the relaxation induced by ET-3 is mediated by
ETB receptor in the smooth muscle
cells of the artery. The endothelium-independent relaxation induced by
ET-3 was not observed in thoracic aortas, pulmonary arteries, renal
arteries, and femoral arteries. Further studies are needed to clarify
the physiological or pathophysiological roles of the
endothelium-independent relaxation mediated by
ETB receptor.
Treatment with a nitric oxide synthase inhibitor, such as
L-NAME and
L-NNA, did not affect the
concentration-dependent relaxation induced by ET-3 in both
endothelium-intact and -denuded rabbit mesenteric arteries.
Furthermore, the ET-3-induced relaxation in endothelium-intact arteries
was not influenced by treatment with OxyHb. It has been reported that
the vasodilatation mediated by nitric oxide is suppressed by OxyHb
treatment (14). In addition, ET-3 did not increase the cGMP content in
the arteries (Table 2). Therefore, it appears that ET-3-induced
relaxation in the arteries is not associated with the nitric oxide-cGMP
pathway. Recently, Wright et al. (35) showed that ET-1 stimulates
PGI2 formation in rat aorta via
activation of ETA receptors in the smooth muscle layer. Thus we next examined the effect of treatment with
a cyclooxygenase inhibitor on the ET-3-induced relaxation of the
arteries. Treatment of the endothelium-denuded arteries with
indomethacin or aspirin completely inhibited the relaxation. Furthermore, ET-3 increased the release of not only
6-keto-PGF1, a stable
metabolite of PGI2, but also
PGE2 from the subendothelial components of the arteries, although the increase of
PGE2 was less than that of
6-keto-PGF1. It is well known
that the vasodilator prostaglandins such as
PGI2 and
PGE2 stimulate the production of
cAMP in vascular smooth muscle (27). We also showed that ET-3 increased
the production of cAMP, which was abolished by treatment with
indomethacin. These results indicate that ET-3 stimulates the
production of vasodilator prostaglandins and subsequently stimulates
cAMP synthesis in endothelium-denuded arteries. Concentration-dependent relaxations induced by PGI2 and
PGE2 were also observed in the endothelium-denuded arteries. Thus our results strongly suggest that
the ET-3-induced relaxation is associated with vasodilator prostaglandins released from subendothelial tissues of the arteries. In
the present study, combined treatment with indomethacin and ET-3
slightly increased the cGMP content compared with that of ET-3,
although indomethacin did not increase the cGMP content of the arteries
treated with vehicle. It is not clear why combined treatment with
indomethacin and ET-3 increased the cGMP content.
To demonstrate the expression of both
ETA and
ETB receptors on rabbit mesenteric
arteries, we next performed the radiolabeled ligand-binding assay using
the quantitative autoradiography technique. Specific
[125I]ET-1 binding
sites were detected on the media and on the perivascular structures of
the arteries as previously reported for human coronary artery (23). The
specific binding of
[125I]ET-1 in the
presence of a high concentration of BQ-123 and the specific binding of
[125I]IRL-1620 were
also observed on these layers and were found to completely disappear on
treatment with BQ-788. Scatchard analysis of these bindings showed that
the Bmax value of
[125I]ET-1 binding in
the presence of BQ-123 was one-tenth of that of
[125I]ET-1 binding and
almost equal to that of
[125I]IRL-1620. These
results demonstrate that not only
ETA receptors but also
ETB receptors are expressed on the
smooth muscle layers of rabbit mesenteric arteries.
Recently, the heterogeneity of the
ETB receptor has been speculated;
the ETB receptor subtype that is
located on vascular smooth muscle and mediates direct vasoconstriction
(ETB2 receptor) appears to be
pharmacologically distinct from the subtype, which is located on the
vascular endothelium and mediates vasorelaxation (ETB1 receptor) (8). The
ETB1 receptor-mediated
vasorelaxation is inhibited by PD-142893, bosentan, or RES-701-1
(5, 12, 33), whereas the ETB2
receptor-mediated vasoconstriction is inhibited by SB-209670 (21).
BQ-788 is an ETB-selective peptide antagonist and appears to inhibit both
ETB1 and
ETB2 receptors with similar
affinity (11). In the present study, the ET-3-induced relaxation in the
endothelium-denuded arteries was sensitive to BQ-788 and PD-142893 but
not BQ-123. From the criteria of classification of the
ETB receptor subtype, our results
indicate that the ET-3-induced relaxation may be mediated by the
ETB1 receptor subtype expressed on
the smooth muscle cells of rabbit mesenteric arteries.
Mizuguchi et al. (17) demonstrated that the multiple functional
ETB receptor subtypes are derived
from the same ETB receptor gene,
because there were no responses of a sarafotoxin S6c-induced relaxation
of the knockout mouse thoracic aorta and sarafotoxin S6c or
IRL-1620-induced contraction of the knockout mouse gastric fundus.
Cheng et al. (4) suggested that alternative RNA splicing contributes to
the regulation of ETB receptor
gene expression. It has also been reported that the EP3 receptor
isoforms of the PGE2 receptor
produced by alternative splicing of mRNA, which generates multiple
protein isoforms from a single gene, couple to different G proteins to
activate different second messenger systems (19). It remains unclear
whether the ETB receptor subtype located in rabbit mesenteric arteries is derived from the same gene
that is expressed on the smooth muscle cells of rabbit pulmonary arteries and induces the vasoconstriction or on the endothelium releasing vasodilator substances (33, 34). Also unknown is which G
protein couples to the endothelium-independent ET-3-induced relaxation.
Further studies are required to elucidate the underlying mechanisms for
the pharmacological heterogeneity of
ETB receptors located in the
vascular smooth muscle layer of rabbit mesenteric arteries.
In this study, we present the first evidence that activation of
ETB receptor induces
vasorelaxation, which is not affected by removal of the endothelium.
This vasorelaxation mediated by smooth muscle
ETB receptor seems to be
associated with the release of vasodilator prostaglandins from the
subendothelial components of rabbit mesenteric arteries.
| |
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: M. Nakajima, Discovery Research
Laboratories II, Shionogi & Co., Ltd., 3-1-1 Futaba-cho,
Toyonaka, Osaka 561-0825, Japan.
Received 17 April 1998; accepted in final form 25 September
1998.
| |
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