Molecular Human Reproduction vol.3 no.12 pp. 1029–1035, 1997
The mechanism of myometrial contractions induced by
endothelin-1 in rat
S.Sakamoto1,3, S.Obayashi1, T.Aso1, J.Sato2, H.Hamasaki2 and H.Azuma2
1Department of Obstetrics and Gynecology, Faculty of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima,
Bunkyo-ku, Tokyo 113, and 2Department of Medicinal Chemistry, Institute for Medical and Dental Engineering, Tokyo
Medical and Dental University, 2-3-10 Surugadai, Kanda, Chiyoda-ku, Tokyo 101, Japan
3To
whom correspondence should be addressed
Experiments were performed to characterize endothelin-1-induced contractions and the role of endothelin
(ET) receptor subtypes in rat myometrium. The binding sites of [125I]-ET-1 were saturable with high affinity.
Scatchard plot analysis revealed that ET-1 binding sites in the myometrium constituted a single population.
The dissociation equilibrium constant (Kd) and the maximum binding sites (Bmax) were determined to
be 48.9 K 3.0 pM and 1364.0 K 210.3 fmol/mg protein respectively. Specific [125I]-ET-1 binding was
inhibited completely by unlabelled ET-1 and Ro 46-2005 (mixed-type ET receptor antagonist), but not fully
(90.7 K 1.4%) by BQ 123 (a selective ETA receptor antagonist), and not at all by RES 701-1 (a selective ETB
receptor antagonist). ET-1 induced myometrial contractions were composed of two types, an increase in
resting tone and rhythmic contractions. These contractions were inhibited by BQ 123 and Ro 46-2005, but
not by RES 701-1. ET-1-induced contractions were greatly reduced in Ca21-free Krebs’ solution. Nifedipine
abolished the rhythmic contractions without affecting the increase in resting tone. These results suggest that
ETA receptors are predominantly localized in rat myometrium and that excitation of ETA receptors evokes
two types of contractions by increasing the cytoplasmic Ca21 concentration.
Key words: ET-1/ETA receptor/ETB receptor/ET receptor antagonist/myometrial contraction
Introduction
Endothelin-1 (ET-1) is the most potent vasoconstrictive peptide
known to date (Yanagisawa et al., 1988). Two other isoforms
are designated ET-2 and ET-3 (Inoue et al., 1989) and all three
isoforms consist of 21 amino acid residues. The actions of
ETs are mediated through excitation of ET receptors which
are classified into two subtypes; the ETA receptor is selective
for ET-1 and ET-2 while the ETB receptor is non-selective for
all three isoforms (Arai et al., 1990; Sakurai et al., 1990).
In addition to its vasoconstrictive properties, ET-1 has
been shown to influence non-vascular smooth muscle tissue,
including rat (Kozuka et al., 1989; Sakata et al., 1989; Sakata
and Karaki, 1992) and human myometrium (Word et al., 1990).
The localization of ET receptors was demonstrated in the
myometrium of rabbit (Maggi et al., 1991) and human
(Breuiller et al., 1994; Bacon et al., 1995). On the basis
of these findings, ET-induced myometrial contractions were
proposed to play a role in the initiation of parturition in rabbit
(Peri et al., 1992), human (Maggi et al., 1994) and rat (Izumi
et al., 1995) and in the control of menstrual bleeding in human
(Ohbuchi et al., 1995).
With regard to the nature of ET-induced myometrial contraction, a qualitative analysis demonstrated that ET caused two
types of response in the rat myometrium; an increase in
resting tone and a rhythmic contraction (Kozuka et al., 1989).
However, sufficient quantitative analysis has not been carried
out.
Thus, the present experiments were carried out to character© European Society for Human Reproduction and Embryology
ize qualitatively and quantitatively the ET-1-induced myometrial contractions of oestrus rat and the ET receptor subtypes
by using different kinds of ET receptor antagonists. Radioligand
receptor binding assays were also performed.
Materials and methods
Reagents
The following reagents were used: ET-1 (human) was purchased from
Protein Research Foundation, Osaka, Japan. Bacitracin, aprotinin,
leupeptin, pepstatin A and bovine serum albumin (BSA; fraction V)
were all obtained from Sigma, St Louis, MO, USA. [125I]-ET-1
(specific activity 2200 Ci/mmol) was purchased from Du Pont-New
England Nuclear, Wilmington, DE, USA. Cyclo (D-Asp-L-Pro-D-ValL-Leu-D-Trp-) (BQ 123), a selective antagonist for ETA receptor
subtype (Ihara et al., 1992) and 4-tert-butyl-N-[6-(2-hydroxyethoxy)5-(3-methoxyphenoxy)-4-pyrimidinyl]-benzenesulphonamide (Ro 462005), a mixed-typed antagonist for ETA and ETB receptors (Clozel
et al., 1993), were synthesized by the Chemical Research Department,
Teikoku Hormone Manufacturing (Tokyo, Japan) and were generous
gifts from the company. Cyclic (Gly1-Asp9) (Gly-Asn-Trp-His-GlyThr-Ala-Pro-Asp-Trp-Phe-Phe-Asn-Tyr-Tyr-Trp) (RES 701-1), a
selective antagonist for ETB receptor subtype (Tanaka et al., 1994)
was a gift from Dr Matsuda, Tokyo Research Laboratories (Kyowa
Hakko Kogyo, Tokyo, Japan).
Animals and tissues
Vaginal smears were taken every morning from female Sprague–
Dawley rats, aged 10–12 weeks. Rats with a confirmed oestrus cycle
1029
S.Sakamoto et al.
were anaesthetized with ether and blood was withdrawn from the
abdominal aorta prior to hysterectomy. Immediately after enucleation,
the uteri were immersed in oxygenated, ice-cold modified Krebs’
solution (NaCl 115.0, KCl 4.7, MgSO4 7H2O 1.2, CaCl2 2H2O 2.5,
KH2PO4 1.2, NaHCO3 25.0 and glucose 10 mM). For measurement
of mechanical responses, uteri were cut into longitudinal strips, 2 mm
in width and 5 mm in length. For radioligand receptor binding assay,
a membrane fraction was prepared from the homogenized uterus after
removing the epithelial layer from the luminal surface with a
surgical knife.
The study was conducted in compliance with the Animal Welfare
Regulation of Tokyo Medical and Dental University and in accordance
with UK legal requirements.
Preparation of crude membrane fraction
The crude membrane fraction was prepared according to the method
described previously (Azuma et al., 1994). In brief, myometrial
tissues were minced with scissors and homogenized in a Polytron at
maximum speed for 20 s to obtain a 25% homogenate in buffer A
(20 mM HEPES, 250 mM sucrose, 5 mM EGTA, 3 mg/ml leupeptin,
2 mg/ml aprotinin, 0.25 mg/ml bacitracin, 3 mg/ml pepstatin A,
pH 7.4). The homogenate was centrifuged at 1200 g for 20 min at
4°C. The supernatant was removed and centrifuged at 80 000 g for
60 min. at 4°C. The resultant pellet was resuspended in buffer A as
a crude membrane fraction and stored at –80°C until use. Protein
concentration was determined with the micro BCA kit (Pierce,
Rockford, IL, USA).
Radioligand receptor binding assay
Radiolabelled ligand used for saturation analysis was [125I]-ET-1.
Crude membrane fraction (20 µl containing 5 µg protein) was added
to 160 µl buffer B (30 mM HEPES, 150 mM NaCl, 5 mM MgCl2,
0.5 mg/ml bacitracin, 1 mg/ml BSA) and 20 µl [125I]-ET-1 at eight
different concentrations of 3.32–350.0 pM and shaken for 120 min
at 25°C. After addition of 3 ml ice-cold buffer B, the mixture was
filtered under reduced pressure through a Whatman GF/B glass-fibre
filter (Whatman, Maidstone, UK). After two washes with 3 ml buffer,
the filters were dried in an oven and transferred to counting tubes.
Radioactivity was determined in a γ counter (Auto-Gamma 800C,
Packard, Meriden, CT, USA). Specific binding was defined as total
binding minus non-specific binding measured in the presence of 125
nM unlabelled ET-1. Displacement of the specific binding
.
of [125I]-ET-1 (45 pM 5. Kd) was performed with ET-1
(3310–10 to 3310–8 M), BQ 123 (10–10 to 10–5 M), RES 701-1 (10–9
to 10–5 M) and Ro 46-2005 (10–8 to 3310–5 M). The concentration
of ligand that gave 50% inhibition of the specific binding of labelled
ET-1 (IC50) was determined by linear regression analysis of the
displacement curve converted through logit transformation.
Measurement of mechanical responses
The mechanical responses were measured according to the methods
described previously (Azuma et al., 1992). A longitudinal uterine
strip was mounted vertically in an organ bath containing 5 ml of
modified Krebs’ solution, continuously bubbled with 95% O2 and
5% CO2 at 37°C. One end of each strip was secured to the bottom
of the organ bath, and the other was attached to a force-displacement
transducer (TB-611T; Nihon Kohden Kogyo Co, Tokyo, Japan).
Isometric changes in tension were recorded on a pen writing oscillograph (R-64; Rikadenki Kogyo Co, Tokyo, Japan). The length of the
strips was adjusted several times until a stable tension of 400 mg
was attained. Before beginning the experiments, strips were allowed
to equilibrate for at least 60 min in the bathing solution and during
this period the bathing solution was replaced every 20 min with fresh
1030
Figure 1. Parameters measured for endothelin (ET)-1-induced
myometrial contractions. The increase in resting tone and the
amplitude of rhythmic contraction were measured at 5, 10, 15 and
20 min after the addition of ET-1. The frequency of rhythmic
contraction was the number occurring between 5 and 15 min.
solution. After 60 min of equilibration, a single concentration of ET1 was applied to one uterine strip and the responses were recorded
for 20 min. In order to construct the concentration–response curves,
the concentration of ET-1 in the organ bath was increased by one
half log unit from 3310–10 to 3310–8 M. For subtyping those
receptors which mediate myometrial contractions, each strip was
incubated in the presence or absence of BQ 123, RES 701-1 or Ro
46-2005. After a 20 min pretreatment with each antagonist, 3310–8
M ET-1 was added to each strip and the changes in developed tension
were recorded for 20 min. Finally, 60 mM KCl was added to obtain
the reference contraction.
ET-1-induced myometrial contractions were assessed by changes
in the resting tone (measured at 5, 10, 15 and 20 min after adding
ET-1), amplitude of rhythmic contraction (maximal contraction minus
basal resting tone, measured at 5, 10, 15 and 20 min after adding
ET-1) and frequency of rhythmic contractions (the number of rhythmic
contractions between 5–15 min after adding ET-1) (Figure 1). Increases
in resting tone and amplitude of rhythmic contractions were expressed
as percentages of 60 mM KCl-induced contractions.
Statistical analysis
All data are presented as means 6 SE. The statistically significant
differences between two means were determined by Student’s t-test.
Differences were considered to be significant when P , 0.05.
Results
Radioligand receptor binding assay
The binding of [125I]-ET-1 was saturable with high affinity.
Scatchard plot analysis revealed that the binding sites of
[125I]-ET-1 constituted a single population. The dissociation
equilibrium constant (Kd) and receptor density (Bmax) values
were determined to be 48.9 6 3.0 pM (n 5 3) and 1364.0 6
210.3 fmol/mg protein (n 5 3) respectively (Figure 2).
As shown in Figure 3, unlabelled ET-1 and Ro 46-2005
.
inhibited the specific [125I]-ET-1 (45 pM 5. Kd) binding in a
concentration-dependent manner respectively. Complete inhibition was attained at a concentration of 3310–8 M of unlabelled
ET-1 and 10–5 M of Ro 46-2005. In contrast, the 125I-ET-1
binding was not fully inhibited with BQ 123 even at the
Characterization of ET-1 induced myometrial contraction
Figure 2. Scatchard plot analysis of [125I]-endothelin (ET)-1
binding to rat myometrial membrane. (A) Binding of [125I]-ET-1
was saturable with high affinity. (B) A single population of binding
sites was demonstrated in the rat myometrium. Kd and Bmax were
determined to be 48.9 6 3.0 pM and 1364.0 6 210.3 fmol/mg
protein respectively.
Figure 4. The concentration–response curve for endothelin (ET)-1
causing: (A) an increase in resting tone at 10 min; (B) an increase
in frequency between 5 and 15 min; and (C) an increase in the
amplitude of rhythnic contractions at 10 min after adding ET-1.
Each point represents the mean value of six experiments. Vertical
bars show SEM. Resting tone was increased by ET-1 at
concentrations ranging from 3310–9 M to 3310–8 M in a
concentration-dependent manner. Rhythmic contractions were
induced at a concentration of 10–9 M ET-1. Frequency of the
rhythmic contraction increased in a concentration-dependent
manner, while amplitude was almost constant from concentrations
of 10–9 M to 10–8 M ET-1.
.
Figure 3. Inhibition of [125I]-endothelin (ET)-1 (45 pM 5. Kd)
binding to rat myometrial membrane by ET-1 (3), BQ 123 (s),
RES 701-1 (u) and Ro 46-2005 (∆). [125I]-ET-1 binding was
inhibited completely by ET-1 and Ro 46-2005, partially (90.7%) by
BQ 123 and was unaffected by RES 701-1. The IC50 values of
unlabelled ET-1, Ro 46-2005 and BQ 123 were 2.0310–10,
1.6310–7 and 9.2310–10 M respectively.
highest concentration of 10–5 M. The binding sites not inhibited
with BQ 123 were calculated to be 9.3 6 1.4% (n 5 3) of
the total specific binding. RES 701-1 at concentrations of
10–8 M to 3310–5 M did not produce a significant inhibition
of the specific [125I]-ET-1 binding. IC50 values of unlabelled
ET-1, BQ 123 and Ro 46-2005 were 2.0310–10 M,
9.2310–10 M and 1.6310–7 M respectively.
1031
S.Sakamoto et al.
Figure 5. Effects of BQ 123 (s), RES 701-1 (u) and Ro 46-2005
(∆) on (A) the increase in resting tone at 10 min (n 5 6), (B) the
increase in frequency of rhythmic contractions between 5 to 15 min
(n 5 6) and (C) the increase in amplitude of rhythmic contractions
at 10 min (n 5 6) after adding 3310–8 M ET-1. Values are mean
6 SE. *P,0.01 compared with corresponding control. Increases in
resting tone were significantly inhibited by treatment with BQ 123
(10–7 to 10–5M) and Ro 46-2005 (3310–6 to 3310–5 M), but were
unaffected by pretreatment with RES 701-1 (3310–7 to 3310–6
M).Frequency of rhythmic contractions was significantly inhibited
by pretreatment with BQ 123 (10–7 to 10–5M) and Ro 46-2005
(3310–6 to 3310–5 M), but were unaffected by pretreatment with
RES 701-1 (3310–7 to 3310–6 M). The amplitude of rhythmic
contractions was hardly affected by pretreatment with BQ 123
(10–6 to 10–5 M), RES 701-1 (3310–7 to 3310–6 M) or Ro 462005 (3310–6 to 3310–5 M).
Mechanical responses to ET-1
Rhythmic contractions were induced at a concentration of
ù10–9 M ET-1, whereas ù3310–9 M ET-1 was required to
increase the resting tone. Figure 4A shows the concentration–
1032
response curve for the increase in resting tone at 10 min after
adding ET-1 at concentrations of 3310–10 to 3310–8 M. Resting
tone was increased by ET-1 in a concentration-dependent
manner. Similar results were obtained when the measurements
were performed at 5, 15 and 20 min after adding ET-1. The
changes in the number of rhythmic contractions (frequency)
between 5 and 15 min after adding ET-1 are shown in Figure
4B. The increase in rhythmic contractions induced by ET-1 was
observed at concentrations ù10–9 M ET-1 and the frequency
increased in a concentration-dependent manner. The amplitude
of rhythmic contractions at 10 min after adding ET-1 was
already 70.0 6 3.6% of KCl-induced contractions at a concentration of 10–9 M ET-1 and only slightly increased at concentrations up to 3310–8 M ET-1 as shown in Figure 4C. Similar
results were obtained when the measurements were performed
at 5, 15 and 20 min after adding ET-1.
Figure 5A shows the effects of ET receptor antagonists on
the increase in resting tone at 10 min after adding 3310–8 M
ET-1. Pretreatment with BQ 123 (10–7 to 10–5 M) as a selective
antagonist for ETA receptor and Ro 46-2005 (3310–6 to
3310–5 M) as a mixed type antagonist for ETA and ETB
receptors greatly inhibited the increase in resting tone. Almost
complete inhibition was attained by 3310–7 M BQ 123 and
3310–5 M Ro 46-2005. In contrast, RES 701-1 as a selective
antagonist for ETB receptors did not produce a significant
inhibition even at the highest concentration of 3310–6 M.
Similar results were obtained when the measurements were
performed at 5, 15 and 20 min after adding ET-1. The increased
frequency of rhythmic contraction caused by 3310–8 M ET-1
was inhibited by the pretreatment with BQ 123 and Ro 462005 in a concentration-dependent manner, but remained
unaffected by the pretreatment with RES 701-1. BQ 123 at
the highest concentration of 10–5 M almost completely inhibited
the increased frequency, which was however, only partially
inhibited by 3310–5 M Ro 46-2005 (Figure 5B). BQ 123, Ro
46-2005 and RES 701-1 failed to modify the increased amplitude of the rhythmic contractions with 3310–8 M ET-1 (Figure
5C). However, the increases in amplitude and frequency
without change in resting tone which had been caused by
lower concentration (10–9 M) of ET-1 were effectively inhibited
by BQ 123 (3310–7 to 3310–5 M) in a concentrationdependent manner.
As stated above, a single addition of 3310–8 M ET-1
produced two types of myometrial contraction, an increase in
resting tone accompanied by an increased rhythmic contraction.
These changes in response to ET-1 were greatly inhibited by
20 min incubation in Ca21-free Krebs’ solution (Figure 6B),
but remained unaffected by 10–5 M indomethacin, 10–6 M
atropine, 10–6 M prazosin and 10–6 M losartan as an angiotensin
II receptor (AT1R) antagonist. In contrast, 3310–7 M nifedipine
as a Ca21-channel blocker clearly inhibited the increases in
frequency as well as amplitude of the rhythmic contraction
without affecting the increase in resting tone (Figure 6C).
Discussion
In this study, we have demonstrated that: (i) ETA receptors are
predominantly localized in rat myometrium; (ii) ET-1 induces
Characterization of ET-1 induced myometrial contraction
Figure 6. Effects of 20 min preincubation in Ca21-free Krebs’
solution (B) and 3310–7 M nifedipine (C) on 3310–8 M endothelin
(ET)-1-induced myometrial contractions. (A) Control responses to
3310–8 M ET-1. (B) Both the rhythmic contractions and the
increase in resting tone were completely inhibited in Ca21-free
Krebs’ solution. (C) In contrast, 3310–7 M nifedipine clearly
inhibited the rhthmic contraction without the increase in resting
tone.
two types of myometrial contraction (an increase in resting
tone and an increase in rhythmic contraction) and the responses
increase in a concentration-dependent manner; (iii) excitation
of ETA receptors evokes these two types of contractions by
increasing the cytoplasmic calcium concentration.
In these experiments, we paid particular attention to the
animal model and to the method for measuring mechanical
reponse. It has been reported that administration of oestradiol,
but not oestradiol plus progesterone, increases the density of
ET receptors in the rabbit myometrium (Maggi et al., 1991).
In rat, the serum oestradiol concentration begins to increase
at the pro-oestrus cycle and reaches a peak at the oestrus
cycle, whereas the serum progesterone concentration is also
elevated in the meta-oestrus and both oestradiol and progesterone concentrations decrease in dioestrus cycle. On the basis of
the rabbit study, the density of ET receptors in the myometrium
appeared to be higher in oestrus cycles than other cycles.
Therefore, rats in the oestrus cycle were used in the present
experiments to characterize ET receptors mediating myometrial
contractions. To determine the mechanical response, a modified
Krebs’ solution was continuously bubbled in an organ bath
with 95% O2 and 5% CO2 at 37°C to maintain oxygen
saturation, since oxygen tension has an effect on smooth muscle
contractility (Bodelsson et al., 1996). A single concentration of
ET-1 was applied to each uterine strip rather than a cumulative
application in order to exclude the influence of an excessive
accumulation of prostaglandins (Izumi et al., 1995) and consequent desensitization to ET-1. However, in previous studies
(Sakata et al., 1992; Bacon et al., 1995), the agent was
applied cumulatively to determine the concentration–response
relationship. In the present experiments, the myometrial contractions caused by ET-1 remained unaffected by indomethacin,
atropine, prazosin or losartan,suggesting that prostanoids,
excitation of muscarinic acetylcholine receptors, α-adrenoceptors and AT1 receptors are unlikely to be involved in inducing
the contractions.
ET-1 produced two types of contractions in rat myometrium,
an increase in resting tone accompanied by an increase in
rhythmic contraction. Nifedipine inhibited the ET-1-induced
rhythmic contractions without affecting the increase in resting
tone. Both types of ET-1-induced contraction were completely
inhibited in Ca21-free solution. It seems probable, therefore,
that both responses were evoked by an increase in the cytoplasmic free Ca21 concentration; the rhythmic contractions
were mediated by the increase in Ca21 influx via voltagedependent Ca21 channels, while the increase in resting tone
was mediated by Ca21 influx via another mechanism. These
speculations seem to be supported by Kozuka et al. (1989)
who reported results similar to our observations. On the other
hand, Khac et al. (1994) reported that an accumulation of
inositol phosphates and inhibition of cyclic AMP generation
coupled with the exitation of ETA receptors may increase
cytoplasmic free Ca21 concentration through activating several
types of Ca21 channels and, in turn, cause two types of
myometrial contractions in rat.
ET-1-induced rhythmic contractions were observed at
10–9 M ET-1, but the increase in resting tone was not observed
until 3319–9 M ET-1. These phenomena were also observed
in oxytocin- and prostaglandin F2α-induced myometrial contractions although the increase in resting tone was small
(S.Sakamoto, unpublished observations). This difference in
threshold concentration between the rhythmic contraction and
the increase in resting tone may be due to different signals
transduction systems after ligand-receptor binding. The detailed
mechanism remains to be investigated.
We examined the ET receptor subtypes mediating myometrial contractions by using antagonists for ET receptors.
Bacon et al. (1995) and Héluy et al. (1995) also studied ET
receptor subtypes by examining mechanical rsponse in the
presence of antagonists for ET receptors and reported that ETA
receptors predominantly mediated the ET-1-induced myometrial contraction in human myometrium. However, they did not
differetiate two types of contraction in the human myometrium.
In rats, ET-1-induced increases in resting tone as well as the
frequency of rhythmic contraction was inhibited by pretreatment with BQ 123 as a selective antagonist for ETA (Ihara
et al., 1992) and Ro 46-2005 as a mixed type antagonist for
ETA and ETB (Clozel et al., 1993) in a concentration-dependent
manner, but not by RES 701-1 as a selective antagonist for
ETB (Tanaka et al., 1994). This suggests that the two types
1033
S.Sakamoto et al.
responses caused by ET-1 in the oestrus rat myometrium are
mediated predominantly through excitation of ETA receptor
subtypes, but not ETB receptor subtypes. Sakata and Karaki
(1992) also reported that ET induced rat myometrial contractions through exitation of ETA receptors since the myometrial
contractions were induced with ET-1, but not with ET-3
except at a high concentration of the peptide. In the present
experiments, BQ 123 inhibited ET-1-induced myometrial contraction more potently than Ro 46-2005, because BQ 123 has
a higher affinity than Ro 46-2005 (IC50 for ETA receptors of
BQ 123 and Ro 46-2005 are 22 and 220 nM respectively).
In order to characterize the ET receptor subtypes localized
in the rat myometrium, we performed radioligand receptor
binding studies using [125I]-ET-1, unlabelled ET-1, BQ 123,
RES 701-1 and Ro 46-2005. [125I]-ET-1 binding was inhibited
by unlabelled ET-1 in a concentration-dependent manner and
complete inhibition was attained at 3310–8 M, indicating
the presence of specific [125I]-ET-1 binding sites in the rat
myometrial membrane. The binding of [125I]-ET-1 was saturable with high affinity. Scatchard plot analysis revealed that
ET-1 binding sites in the myometrium constituted a single
population. On the basis of the inhibitory mode of BQ 123, it
is suggested that the specific [125I]-ET-1 binding sites are
composed of two components, BQ 123-sensitive ETA receptors
and BQ 123-resistant binding sites. The former occupied 90.7
6 1.4% (n 5 3) of the total [125I]-ET-1 binding sites (Figure 3).
It has been reported that both ETA and ETB receptors are
localized in the human (Bacon et al., 1995) and rabbit (Maggi
et al., 1991) and sheep (Riley et al., 1995) myometrium.
However, whether or not the remaining 9.3% of the total
binding sites can be classified as ETB is not clear, since RES
701-1 at the highest concentration used did not produce a
significant inhibition of the specific [125I]-ET-1 binding,
whereas binding was completely inhibited by Ro 46-2005.
Therefore, it cannot be excluded that not only ETB but also
putative non-ETA/non-ETB receptors may be involved in the
BQ 123-resistant binding sites. Indeed, Azuma et al. (1995)
suggested that Ro 46-2005 antagonized not only ETA and ETB
receptors, but also putative non-ETA/non-ETB receptors in the
vascular smooth muscle cell membrane. Bacon et al. (1995)
demonstrated that ETA receptors predominate (79% of the
total) in mediating human myometrial contractions, the
remaining 21% of receptors being classified as ETB. The
difference in size of ETA receptor population between rats
(90.7%) and human (71%) may reflect the difference between
oestrus cycles and menstrual cycles. In as much as only two
mammalian ET receptors have been isolated, cloned and
expressed to date, the existence of additional ET receptors has
been postulated based on data not conforming to the existing
characterization of either ETA and ETB receptors. In addition,
evidence is now accumulating to suggest the presence of
multiple subtypes of ETB receptors (Douglas et al., 1994).
Further investigations are in progress in our laboratories to
characterize the BQ 123-resistant [125I]-ET-1 binding sites and
to determine the physiological role of these binding sites in
the myometrium.
In conclusion, ET-1 induced two types of myometrial
contractions, an increase in resting tone and rhythmic contrac1034
tions. Both types of contraction were mediated through excitation of ETA receptors which were the predominant population
(90.7%) in oestrus rat myometrium. The remaining 9.3% of
ET receptors may belong to the ETB type and/or a putative nonETA/non-ETB type. The physiological roles of these receptors in
myometrium remain to be investigated.
Acknowledgements
This study was supported by Grants-in-Aid for Scientific Research
(08457436) from the Ministry of Education, Japan.
References
Arai, H., Hori, S., Aramori, I. et al. (1990) Cloning and expression of a
cDNA encoding an endothelin receptor. Nature, 348, 730–732.
Azuma, H., Niimi, Y. and Hamasaki, H. (1992) Prevention of intimal thickening
after endothelial removal by a nonpeptide angiotensin II receptor antagonist,
losartan. Br. J. Pharmacol., 106, 665–671.
Azuma, H., Hamasaki, H., Niimi, Y. et al. (1994) Role of endothelin-1 in
neointima formation after endothelial removal in rabbit carotid arteries.
Am. J. Physiol., 267, H2259–H2267.
Azuma, H., Hamasaki, H., Sato, J. et al. (1995) Different localization of ETA
and ETB receptors in the hyperplastic vascular wall. J. Cardiovasc.
Pharmacol., 25, 802–809.
Bacon, C.R., Morrison, J.J., O’Reilly, G. et al. (1995) ETA and ETB endothelin
receptors in human myometrium characterized by the subtype selective
ligands BQ123, BQ3020, FR139317 and PD151242. J. Endocrinol., 144,
127–134.
Bodelsson, G., Laursen, M. and Stjernquist, M. (1996) Oxygen promotes
contraction by endothelin-1 in human umbilical artery. Hum. Reprod., 11,
2767–2768.
Breuiller, F.M., Heluy, V., Fournier, T. and Ferre, F. (1994) Endothelin
receptors: binding and phosphoinositide breakdown in human myometrium.
J. Pharmacol. Exp. Therap., 270, 973–978.
Clozel, M., Breu, V., Burri, K. et al. (1993) Pathophysiological role of
endothelin revealed by the first orally active endothelin receptor antagonist.
Nature, 365, 759–761.
Douglas, S.A., Meek, T.D. and Ohlstein, E.H. (1994) Novel endothelin
receptor antagonists welcome a new era in endothelin biology. Trends
Pharmacol. Sci., 15, 313–316.
Héluy, V., Germain, G., Fournier, T. et al. (1995) Endothelin ETA receptors
mediate human uterine smooth muscle contraction. Eur. J. Pharmacol.,
285, 89–94.
Ihara, M., Noguchi, K., Saeki, T. et al. (1992) Biological profiles of highly
potent novel endothelin antagonists selective for the ETA receptor. Life
Sci., 50, 247–255.
Inoue, A., Yanagisawa, M., Kimura, S. et al. (1989) The human endothelin
family: three structurally and pharmacologically distinct isopeptides
predicted by three separate genes. Proc. Natl. Acad. Sci. USA, 86,
2863–2867.
Izumi, H., Byam, S.M. and Garfield, R.E. (1995) Gestational changes in
oxytocin- and endothelin-1-induced contractility of pregnant rat
myometrium. Eur. J. Pharmacol., 278, 187–194.
Khac, L.D., Naze, S. and Harbon, S. (1994) Endothelin receptor type A
signals both the accumulation of inositol phosphates and the inhibition of
cyclic AMP generation in rat myometrium: stimulation and desensitization.
Mol. Pharmacol., 46, 485–494.
Kozuka, M., Ito, T., Hirose, S. et al. (1989) Endothelin induces two types of
contractions of rat uterus: phasic contractions by way of voltage-dependent
calcium channels and developing contractions through a second type of
calcium channels. Biochem. Biophys. Res. Comm., 159, 317–323.
Maggi, M., Vannelli, G.B., Peri, A. et al. (1991) Immunolocalization, binding,
and biological activity of endothelin in rabbit uterus: effect of ovarian
steroids. Am. J. Physiol., 260, E292–E305.
Maggi, M., Vannelli, G.B., Fantoni, G. et al. (1994) Endothelin in the human
uterus during pregnancy. J. Endocrinol., 142, 385–396.
Ohbuchi, H., Nagai, K., Yamaguchi, M. et al. (1995) Endothelin-1 and big
endothelin-1 increase in human endometrium during menstruation. Am. J.
Obstet. Gynecol., 173, 1483–1490.
Peri, A., Vannelli, G.B., Fantoni, G. et al. (1992) Endothelin in rabbit uterus
during pregnancy Am. J. Physiol., 263, E158–E167.
Characterization of ET-1 induced myometrial contraction
Riley, S.C., Findlay, J.K. and Salamonsen, L.A. (1995) Endothelin-1 and
endothelin receptors are present in the sheep uterus and conceptus at
implantation. J. Endocrinol., 147, 235–244.
Sakata, K. and Karaki, H. (1992) Effects of endothelin on cytosolic Ca21
level and mechanical activity in rat uterine smooth muscle. Eur. J.
Pharmacol., 221, 9–15.
Sakata, K., Ozaki, H., Kwon, S.C. and Karaki, H. (1989) Effects of endothelin
on the mechanical activity and cytosolic calcium level of various types of
smooth muscle. Br. J. Pharmacol., 98, 483–492.
Sakurai, T., Yanagisawa, M., Takuwa, Y. et al. (1990) Non-isopeptide-selective
subtype of the endothelin receptor. Nature, 348, 732–735.
Tanaka, T., Tsukada, E., Nozawa, M. et al. (1994) RES-701–1, a novel, potent,
endothelin type B receptor-selective antagonist of microbial origin. Mol.
Pharmacol., 45, 724–730.
Word, R.A., Kamm, K.E., Stull, J.T. and Casey, M.L. (1990) Endothelin
increases cytoplasmic calcium and myosin phosphorylation in human
myometrium. Am. J. Obstet. Gynecol., 162, 1103–1108.
Yanagisawa, M., Kurihara, H., Kimura, S. et al. (1988) A novel potent
vasoconstrictor peptide produced by vascular endothelial cells. Nature, 332,
411–455.
Received on June 23, 1997; accepted on September 23, 1997
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