BIOLOGY
OF
46,
REPRODUCTION
Effects
392-400
(1992)
of Prostanoids
on the
Rat’s
Myometrium
D. J. CRANKSHAW Department
of Obstetrics
and
and Gynecology, Hamilton, Ontario,
In Vitro
during
Pregnancy1
V. GASPAR
Health Sciences Canada L8N
Centre, 3Z5
McMaster
Universiiy
ABSTRACT The
objectives
metrium
PG
of this
vitro
in
(prostaglandin)F,,,
Days
10,
circular
were
study
to a number
the
PGD,,
15,
18, 20,
strips
were
21,
10;
thereafter
potency
than
at Day
10. In contrast,
fell
PGF2,,
Responses value
on Day
for PGE2
were
no changes
there
to PGE2
and
during
PGF,,,
pregnancy
sthps
18 and
then
and
a high
gradually
in potency
the period
response
studied
were
U-46619 but
until
maximal
to
investigated
on
it
(longitudinal
the
that
hypotheses
these
prostanoids
act at different
lower
still
was
or a slight
muscle)
Both ioprost and U-46619 maintained a low potency throughout is probably not a target for PGI2 or TxA2 and that the different support
and
on Day
response
22, when
Day
myo-
longitudinal
between
low
of study
of the Responses
separately.
differences
potency
increased
over
contractile
were
A2-mimetic
prostanoid-dependent, with
on the
rat
(Tx)
thromboxane
patterns,
similar
to a minimum
increase between Days 15 and 21 (circular muscle). We conclude that the pregnant rat’s myometrium responses
were
in the oriented
circularly
the
and
produced
PGD,
of pregnancy and
iloprost,
PGI2-miznetic
and
effects
the
Longitudinally
22 of pregnancy.
and
small.
to determine
of prostanoids.
pregnancy.
patterns
of
sites within
the
myometrium.
INTRODUCTION Prostanoids rition muscle
are
thought
to play
[11, in part through their direct of the uterus [2,31. Several
a major
role
in partu-
effects on the smooth observations have led
analogs and
effectively
induce
labor
the horse [8] but not The role that prostanoids
close
to term
in vitro
suggest
that
in the
it
becomes
unclear. Some on longitudinal more
[7]
membrane an increase on
depolarization. in sensitivity
longitudinal
pared
to Day
muscle 19 pregnant
Accepted
October
18,
Received
August
1, 1991.
needed discharges
to
produce without
rats,
Day
22
although
pregnant the
rats maximum
however,
circular
on Day [10] was
21 than unable
of PGEI, PGE2, were successful.
or aspirin-treated
these agents produced some metrium through mechanisms prostanoid synthesis. There are no reliable data prostanoids
an inmarked
comef-
the maximum muscle from muscle
reDay was
on Day 17. to induce par-
or PGF28,, Furthermore,
although intra-
by prostanoid infusions than they were on Day that the characteristic be brought about by a
interaction between a number of prostanoids. they interpreted the failure of PGF28, to restore electrical activity to muscle strips isolated from in-
domethacin-
as term
Reiner and Marshall [12] found to the mechanical effects of PGF2,,, from
rats;
more sensitive intact rat, Fuchs with infusions of oxytocin
complex However, normal
approaches. Thus Kuriyama and Suzuki [11] investigated electrical but not mechanical responses to PGE2 and found that between Day 15 and parturition there was a marked decrease in the concentration crease in the frequency of burst
21 pregnant
50% of longitudinal
uterine pressure changes induced were less on Day 22 of pregnancy 21. Anderson et al. [14] recognized myometrial changes at term may
invesmus-
sensitive
that produces for PGF28, on
turition infusions
its
pig
concentration sponse) values significantly In the
in the sheep [9] or the rat [10]. play in stimulating uterine ac-
tivity at term in the rat is particularly tigations of the effects of prostanoids cle
[3]. to prostanoids; (PG) F28, or
similar on both days. In contrast, Tuross et no significant differences in EC50 (effective
15, 17, or
to the formulation of this hypothesis: the prostanoids are potent modifiers of myometrial contractility [4]; labor can be induced by prostanoid administration [5]; parturition can be delayed by administration of prostanoid synthesis inhibitors [6]; and prostanoid concentrations in the uterus undergo acute changes around the time of parturition Not all species respond in the same way thus it has been claimed that prostaglandin
fect appeared al. [13] found
during
pregnant
rats
of their other
effects than
concerning
pregnancy,
as evidence
the
although
that
on the inhibition effects
some
myoof
of other
evidence
suggests that they may play an important Dubin et al. [15] have presented evidence rat’s uterus produces substantial amounts
role. For instance, that the pregnant of thromboxane
(Tx) A2, which they claim ity. In the lower segment
myometrial human
could regulate of the pregnant
activuterus,
the stable Tx-mimetic U-44069 has been shown to be a potent uterotonic [16], whereas in the nonpregnant rat’s uterus a similar compound U-46619 is almost ineffective [17]. Similarly, the pregnant rat’s uterus produces large amounts
1991.
‘This work was supported by an operating grant from the MRC of Canada. It was presented in part at the Philippe Laudat Conference on the Biochemistry and Pharmacology of the Myometrium, Alsace, France, September 30-October 4, 1990. tCorrespondence: DJ Crankshaw, McMaster University, Dept of Obstetrics & Gynecology, 1200 Main St. West, Hamilton, Ontario, Canada LEN 3Z5. FAX: (416)5770471.
of PGI2 are potent ment 392
[18];
ineffective
and
despite
on the
the
nonpregnant
fact
that rat’s
stable uterus
PGI2
mimetics
[17], they
inhibitors of pregnant human lower uterine activity [16]. Three major questions stimulated
are segthe
PROSTANOIDS present
studs’.
sitivity metics
First,
are
there
drastic
of the pregnant rat’s uterus that might indicate that the
important ond, when
regulators measured
responses
of the
uterus
to
in the
sen-
to PGI2- and TxA2-minatural compounds are
of contractility under similar rat’s
changes
PREGNANT
AND
during pregnancy? Seccircumstances, are the
PGE2
and
PGF2C, different,
as circumstantial evidence would suggest [11, 13], thus perhaps indicating different roles for the two prostanoids? Finally, can the possible regulation of prostanoid responses during pregnancy give any clues to the question whether different prostanoids To find answers
act to
at different sites [17]? these questions, we examined
the
MYOMETRIUM
out, the tension was allowed justed to 10 mN if necessary), iment
proper
was
Measurement
and
storage
periods. of these
of the
square centimetre Spontaneous
second
half
of
AND
Animals Timed-pregnant River facilities ad
sperm housed
rats
were
St. Constant,
at 22#{176}C on
water
libitum.
a 12L: 12D
The
suppliers
purchased
from
PQ.
They
were
cycle
and
allowed
designated
Charles
kept
the
Strips
in our
food
and
morning
that
were found in the vagina after females had been overnight with a male rat as Day I of pregnancy.
When they
Wistar
Laboratories,
animals
were
normally
allowed
delivered
to reach
on
the
term
morning
in our
facilities,
of Day
in
used
ments,
only
of each used
The
Animals were killed ignated day of pregnancy.
by a blow to the head on the The entire uterus containing
products
was
(95% 02: temperature.
C02) physiological The PSS had the
5%
KC1, 4.6; MgSO4, 1.16; NaHC03, 21.9; glucose, Uteri removing
removed
were the
trimmed fetuses,
and
placed
direction. No attempt and circular muscle
dometrium. taken from tied
with
organ resting was
Most often sites adjacent silk
longitudinal to different and
monitored
through
to MA).
mm
before
that
failed and
a Grass Tissues
suspended
being to respond where
a Grass
polygraph allowed
challenged
with
with possible
vertically
isometric
strips strips
were were
90
mM
initial Force
transducer
KC1. Any to .KC1 was
The
taken
from
each per animals
each
strip. by constructing agent as we have
activity In
was
were
not
dose-response
tested
on
each
strip.
(n) refers to both the of strips since, although
were
taken
from
each
used
experiIn all
number of four strips
animal,
each
was
purpose.
sessed
Anal’’sis significance using
of differences
Student’s
test [19] using p < Values are expressed the
t-test 0.05
between
combined
as the as means
values
with
was
a multiple
as-
range
cut-off point for significance. ± the standard deviation
of
mean.
Materials except
E. Schillinger, Schering Chemical, Ann Arbor, as previously described from
Sigma
chemicals
Chemical were
from
iloprost,
which
was
AG, were obtained MI. They were prepared [17]. Indomethacin Co., BDH,
TABLE 1. Spontaneously developed circularly cut strips of myometrium
St.
Louis,
Toronto,
MO,
a gift from
Dr.
from Cayman and stored was obtained and
all
other
ON.
tension in longitudinally from pregnant rats.
and
in a 10-ml
(Grass Instruments, to equilibrate for
a contraction replaced.
Four in the
PSS at 37#{176}C. An on each strip.
FT-03
7-D were
midhorn. and four
and circular fetuses. The
for
spontaneous
prostanoid
a different
All prostanoids
made to separate lonor to remove the en-
bath containing oxygenated force of 10 mN was exerted
connected Quincy,
carded
thread
was layers
115.5;
tissue; without cut from over
the back of a fetus implanted approximately strips were cut in the longitudinal direction circular gitudinal
at room (mM):
1.16; NaCI, 0.01.
of fat and connective strips of uterus were
desthe
in gassed
salt solution (PSS) following composition
CaC12, 2.5; NaH2PO4, 11.1; indomethacin,
10-mm
22.
Preparation
of conception
strips
were determined curves to each
number number
orientation for
over
was determined by taking Activity was expressed as a
experiments. one
at 2 Hz
activity present during in terms of Newtons
record
to assess
cases, the given animals and the
quantified as we digitalization
[17]. In most experiments there was and nine concentrations of prostanoid, of these experiments equal to those in activity was assessed.
dose-response
Statistical Tissue
first
of prostanoids dose-response
described previously one control period making the length which spontaneous
METHODS
output
of pregnancy in succession.
of the
Effects cumulative MATERIALS
amplifier
(N/cm2). activity of muscle
percentage
the
(readexper-
begun.
The mean contractile periods was expressed
strips of pregnancy.
throughout
to baseline mm the
is
activity of the strips was earlier [17] b simultaneous
effects of PGF78,, PGE2, PGD2, iloprost, and U-46619 on the contractility of both longitudinally and circularly oriented myometrium
to decline and after
of Contractility
The contractile have described
at different stages ten such records
rat
393
KCI was
20 strip dis-
washed
Day 10 15 18 20 21 22
of pregnancy
Longitudi 0.9 1.7 1.2 1.7 1.0 1.1
nal strips ± ± ± ± ± ±
Circula
0.3 0.6 0.5 0.7 0.3 0.3
Tissue strips were set up and spontaneously developed termined as described in Materials and Methods. Values are means ± SD from 10 animals in each case;
0.9 1.0 1.3 1.0 0.9 0.9 tension units
r strips ± ± ± ± ± ±
0.5 0.4 0.4 0.3 0.3 0.5
was
de-
are N/cm2.
394
CRANKSHAW CIRCULAR
AND
LONGITUDINAL
Daytl
GASPAR
tation of the strips. There were no marked differences between days for the same orientation or between tations for the same day.
DaytO
250
In general, mained fairly
150
or of the 50
once constant
orientation
of the
of the experiment. This (right panel, longitudinal DayI5
DaylS
250
Absolute
150
in the two muscle Day 22 of pregnancy
‘LI
50
-J
4 >
-J
nflflli. DayiB
0
values
DayIO
I-
z
0 50 4 U)
strips that
UHfldfl
250
150
50
1
I0 4
noon”.
duud.
Oay2i
Day2l
250
I-
z 0 C)
150
0ay22
90
10
TIME
30
50
70
90
expressed as a percentage of the activity in the n = 10-12, and errors are standard deviations
of the means. The elapsed time between the and of one measurement riod and the beginning of the next was less than 1 mm. Right-panel, gitudinally oriented strips; left-panel, circularly oriented strips.
pelon-
1 shows
veloped
by control
periment
proper,
the
values strips
against
of the during the
spontaneous the
day
on
PGE2,
prostanoid Iloprost
first
developed
was
10 mm
of pregnancy
force
circularly
consistent greater
oriented
observation tension. Re-
small.
activity
de-
of the and
The
sensitivity
maximum tension Days 18 and 20 for in responses
Day
10 and
of Day
de-
20; therethe values
development PGF28, and
to PGE2
subsequently
10. Maximum
making
declined tension
the
general
(Fig.
on
4) was
to values
generated
pattern
less exaggerated. (Fig. 5) was excitatory
TABLE 2. Effects of prostanoids from Day 22 of pregnant rats.
Agonist PGF2 PGD2 PGE2 lloprost U-46619
RESULTS Table
developed
and
close
on Day
ex-
orien-
± ± ± ± ±
0.3 0.5 0.5 0.4 0.4
with
during
in circular The pattern
on longitudinal
strips
10
preg-
strips on of change
Maximal force (N/cm2) 1.6 ± 0.7 3.5 ± 2.7 2.4 ± 1.5 2.7 ± 2.0 3.6 ± 0.2
0.63
2.5 0.5 1.6 16
Negative log of the molar concentration giving ± the standard deviation of the mean. bMicromOlar concentration giving a half-maximal
this
of myometrium
EC (M)’
pD2 6.2 5.6 6.3 5.8 4.8
of change
at all times
nancy, although the tension developed Days 10 and 15 was extremely low.
(mm)
FIG. 1. Spontaneous contractile activity in strips of myometrium isolated from pregnant rats. Activity was measured over 10-mm periods as described in Materials and Methods. The height of each bar represents the activity in that time period first (control) time period;
maximum
longitudinal
of Day 10). In contrast, reached its peak between Day 22 for PGD2. The pattern of change
for 70
duration
by PGE2 was higher in both longitudinal (2-fold and 1.4fold respectively) and circular strips (4.1-fold and 4.5-fold respectively) than that generated by either PGF2C. or PGD2. However, the absolute maximum values were generally lower
Day22
50 50
between
tension
to those
150
30
and
creased, reaching a nadir between Days 18 and after it slowly increased (though it never reached
than
250
10
the
quite different. In longitudinal strips there were no significant changes in sensitivity throughout the study period, whereas in circular strips sensitivity was greater on Day 15
ui
50
for
layers in response to the prostanoids on are shown in Tables 2 and 3, respec-
were minimal except for the longitudinal strips developed
imum
>.
I> I.C.) 4 uJ
strips,
sponses to PGF2Q and PGD2 (Figs. 2 and 3) changed with very similar patterns. In early pregnancy, the rat’s uterus was highly sensitive to these two prostanoids, but the max-
Day2O
Day20
4
muscle
developed it reday of pregnancy
is shown graphically in Figure 1 strips; left panel, circular strips).
for sensitivity
Differences 150
U-
activity of the
tively. Changes in sensitivity and maximum developed forces of longitudinal and circular muscle strips to the prostanoids PGF28,, PGD2, PGE2, iloprost, and U-46619 with advancing pregnancy were complex and are represented in Figures 2-6, respectively.
250
0 0
spontaneous irrespective
either orien-
a half-maximal response.
response
PROSTANOIDS of sensitivity to iloprost resembled major exception: in both longitudinal there and
was
a significant
22. The Tx-mimetic
on
strips
U-46619
of both
be detected nor maximal
drop
(Fig.
between
6) had no
Day 18, changed
PREGNANT
a very
low
significant
whereafter significantly.
Days
could
sensitivity
uterus factors:
to prostanoids the uterus
develops
spontaneous
processes
are
[15]. In our performing
are itself
complex,
since
hampered produces
by at least prostanoids, activity.
by the the in the has been tissues
of the
duration
two and
major also
it
of
EC 6.0 ± 0.4 5.3 ± 0.2 6.2 ± 0.6 5.6 ± 0.5
1.0 5.0
U-46619
4.9
spontaneous
activity
of the
log
the standard
molar
1.3 ± 0.3 2.2 ± 1.2 1.1 ± 0.7 1.3 ± 1.2 1.3 ± 0.3
2.5 12.6 concentration
deviation
of the
concentration
giving
pregnancy by
prostanoid problem
in the
sponse
experiments
giving
a half-maximal
a half-maximal
response.
of the
difficult,
is
presence
1 are
since
spontaneous
activity
different
of tissues
obtained
shown
on
those
reported
from
Table
by others.
period measured in a stepwise
0
z
2
22 DAY
OF
PREGNANCY
OF
16 18 PREGNANCY
4
z
0
3
a, 2
Ui
I-
01
a
2
x
0 14 DAY
FIG. 2. Effects of nancy. The solid bars pD2 is the negative log is the maximal tension different from Day 22
OF
16 18 PREGNANCY
20
22
12
14 DAY
in this
1 and
Figure
Dubin
et al. found dur-
after a 15-mm equilibration fashion from Day 15 to Day
O IJJ I-
the
used
oriented strips only and activity, i.e. activity occurring
z a
presents
baseline against which an accurate description
profile
[15] studied longitudinally that the initial contractile ing a 15-mm time, increased
essentially
it
PGF2
12
response
mean.
with a constantly moving responses. Therefore,
study was necessary. The results we
of mndomethacin has been reported to be a time-dependent phenomenon [15]. Changing spontaneous activity over time within a given tissue makes the interpretation of dose-re-
10
force
(N/cm2)
0.63
± 0.1
from
Maximum
PGF2 PGD2 PGE2 lloprost
observers to measure
these
first problem was treated presence of a concentration shown to inhibit [15]. The second
of myometrium
pregnant
Both of the
strips
(p.M)b
bMicromolar
responsiveness
contractile
influenced experiments, all studies
of mndomethacin that production in similar more
the
on circular
pD2
‘Negative
DISCUSSION to describe
of prostanoids rats.
Agonist
±
Attempts
395
21
potency
effects
neither
MYOMETRIUM
TABLE 3. Effects Day 22 of pregnant
of PGE2 with one and circular strips,
in sensitivity
orientations:
before tension
that
AND
20
PGF2 on the contractile activity of isolated strips of pregnant rat’s myometrium during pregrepresent longitudinally oriented strips; the open bars represent circularly oriented strips. The of the molar concentration of prostanoid producing a half-maximal response. Maximal tension developed in response to the prostanoid; units are N/cm2. Values marked “+“ are significantly of pregnancy, Values marked “*“ are significantly different from Day 10 of pregnancy.
396
CRANKSHAW
AND
GASPAR
PGD2 4
10
z
8
0 U)
3
z
w I-
2
fHii
:
4
1
0 10
12
14
DAY
OF
16
18
20
22
10
PREGNANCY
12
14
DAY
OF
16
18
20
22
PREGNANCY
4 2 0 C’, 2
w
*
I-
01
a
0. *
4
0 10
FIG. 3. nancy. The
12
14
DAY
OF
16
18
20
22
n
I-I
10
PREGNANCY
Effects of PGD2 on the contractile solid bars represent longitudinally
activity oriented
Fi
t t
of isolated strips strips; the open
12
14
DAY
OF
16
18
20
22
PREGNANCY
of pregnant rat’s myometrium during pregbars represent circularly oriented strips. The
P02 is the negative log of the molar concentration of prostanoid producing a half-maximal response. Maximal tension is the maximal tension developed in response to the prostanoid; units are N/cm2. Values marked “+“ are significantly different from Day 22 of pregnancy; values marked ““ are significantly different from Day 10 of pregnancy.
22 of pregnancy.
In our
ferences in spontaneous 10-mm interval, which was suspended in the observed
a steady
decline
case,
there
were
no significant
dif-
activities measured during the first began about 45 mm after the tissue bath. Furthermore, Dubin et al. [15] in spontaneous
activity
over
a 60-
therefore surprising to find that in response to the prostanoids there were not any significant differences between the two layers. Tuross et al. [13] stated that “it is preferable to isolate the two muscle layers as completely as possible,
21 for lonWe cannot
since the layers are electrically coupled and the contractions of one layer are synchronized with those of the other, especially at term.” We chose not to separate the layers; our rationale for this was that the interaction between the layers is probably of physiological significance and that our in-
explain the difference between the two studies since, except for the strain of rats used (Sprague-Dawley versus Wistar) the conditions seem to be identical. Nevertheless, these
terest was in how the different layers behave when they are coupled. Furthermore, what we believe to be important physiological differences between the two layers can be
results do indicate that our method of assessing responsiveness was not compromised by fluctuations in baseline. There are a number of circumstances under which longitudinal and circular muscle of the rat’s myometrium
demonstrated when contact between them is unperturbed [21]. Similar logic prompted our decision to leave the dometrium intact. The pattern of responsiveness of the rat’s myometrium
behave quite differently. For instance, the relationship between mechanical activity and the underlying electrophysiological events is markedly different between the two layers [14]: during pregnancy, the two layers react differ-
during pregnancy pare Figs. 2-6). seen, we Tuross
ently
tO
mm period for most days of pregnancy, whereas our observations showed activity to remain relatively constant. There was a greater variability between tissues gitudinal strips and on Day 22 for circular
to catecholammnes
[20]
and
on Day strips.
to oxytocin
[13, 21]. It was
PGF2a
is clearly prostanoid-dependent Because of the complexities of the
will discuss et al. [13] on
Days
each studied 15,
prostanoid in vitro
17, and
21
in turn. myometrial in a colony
en-
(comchanges responses
of rats
that
PROSTANOIDS
AND PREGNANT
MYOMETRIUM
397
PGE2
2
0 U) 2 Ui
01
I-.
a a
4
10
12
14 DAY
16 18 PREGNANCY
OF
;
20
22
t
1:
4
2
6
3.
0 a, 2
5
Ui
01
I-
0
4
a
2
3 4
2
1#{149}
H
1
.1
-
0-
10
12
14
DAY FIG. 4. Effects of nancy. The solid bars pD2 is the negative log is the maximal tension different from Day 22
normally and
delivered
some
ours.
on
notable
They
found
that
ogous
the
muscle between
to those
Days
(0.63
and
pM).
12
14
DAY
16
OF
some
findings
to PGF2U
15 and
21; these
was
right
and no anal-
panel).
Sim-
0.83 j.&.M in longitudinal and are close to those we obtained
responses
to PGF2a changes
(compare they tween
between upper
found Days
to be
on
Day
21
fraction
and
15 and
[13]
circular
muscle
The
lower 21.
found only
magnitude
longitudinal
a progressive
a change within the
number of receptors in coupling between
right
on
Days
of responses and panels
increase It is possible
in
circular of Fig.
21.
to PGF2a muscle
circular that
17 and
strips
muscle the
differences
time. nancy on
2), whereas be-
role
nomena
in the target
Day
any of these
22 (Fig. throughout
panels).
the
later
sites
whole
over change.
differ-
with
this
for PGF2U
rat myometrium time
Day was
15.
Al-
complex,
response of the myobe regulated; among
biotransformation rate tissue; (2) a change
processes
binding
the
a significant
binding
from
is almost
2, left
between
demonstrated
in the target the receptor
observed
by
used.
at which the in vitro might
The increase in PGF2a [22] is consistent with
correlation sponse
that we
explained
compared
of changes significant
always
be
[22]
of pregnancy
though the pattern this was the only
differences. were
graded
22
of high-affinity
membrane
significant to prostanoids
et al.
in the
in
late
number
in this case both In our study,
We
a reduction
in a crude
can
Hertelendy
in the
(1)
Tuross
20
preparations
these are prostanoid
whereas
found
increase
studies
muscle
are many sites to prostanoids
to be similar
et al. [13]
two
in the
Moln#{225}rand
was are
the
ences
greatest
there results
2 (upper
between
similarities
their
17, although
layers showed the responses
18
PREGNANCY
There metrium
found
Tuross
10
22
sensitivity of both muscle layers between Days 17 and 21, but the changes were significant only in the circular layer. We made similar observations between Days 18 and 22, but
graded,
1.0
are
Day
and
20
PGE2 on the contractile activity of isolated strips of pregnant rats myometrium during pregrepresent longitudinally oriented strips; the open bars represent circularly oriented strips. The of the molar concentration of prostanoid producing a half-maximal response. Maximal tension developed in response to the prostanoid; units are N/cm2. Values marked “+“ are significantly of pregnancy; values marked ““ are significantly different from Day 10 of pregnancy.
between
in Figure
ilarly, EC50 values of 0.36 circular muscle respectively
18
PREGNANCY
response
on
shown
OF
21. There
differences
in longitudinal difference
Day
16
of the in the
tissue; and (3) a change and the final response. might
completely
play
in the
unknown
pheat this
binding sites at term of pregthe increase in sensitivity seen However, site part
number
there and
of pregnancy.
is no
direct
in vitro
re-
CRANKSHAW
398
AND GASPAR
ILOPROST 4
2
3
0 In 2
a
Ui
01
I2
a
4
0 10
12
14
DAY
16
OF
18
20
22
PREGNANCY
8
4
7 -
-
2
6
0
-
3
a) 5 01
2
__
3
0
‘t
10
p
effects
of PGD2
T
14 DAY OF
r
7
r
%
16 18 PREGNANCY
22
not reto
in vitro
3). On the basis we observed
changes in receptors or post-receptor to changes in biotransformation)
this
of the tenuous are related to
mechanisms suggests
that
(i.e., not PGF2U
and PGD2 act at the same site in the rat’s myomethum. There is now compelling evidence to suggest that the major prostanoids of the 2-series PGD2, PGE2, PGF2,, PGI2, and TxA2
respectively.
the primary that have There
ligand at one been designated is a crossover
tanoid and receptor with a lower potency there
is a wide
of a series of prostanoid DP, EP, FP, IP, and of activity
in distribution
ceptors within different tissues (for view see Coleman et a!. [23]). Evidence tention pregnant
between
(e.g., PGE2 can act at the than PGD2, the primary
diversity
of the
contraction agonists are
TP
pros-
various
re-
a comprehensive supporting our
recon-
of smooth ineffective
site the
smooth sults). ceptors occurring
OF
I
I
I
16 18 PREGNANCY
20
in the DP re-
muscles [23]. on rat uterine
muscle (Crankshaw These points argue in the
rat
and against
22
myometrium.
prostanoids
Gaspar, unpublished the presence of DP Moreover,
tested,
PGD2
only to PGF2a in systems containing ceptor [23]; in these systems, PGD2 of PGF2a. The nature of the different from those
rere-
of all naturally
is second
in
potency
exclusively acts as a less
the FP repotent an-
changes in response to POE2 to PGF2a and PGD2. There
are clearly were no
alog
significant although
DP receptor ligand), and
that PGF2a and PGD2 act at the same rat’s uterus is as follows. Action at
ceptor tends to inhibit Selective DP receptor
14 DAY
have
are each receptors
12
iloprost on the contractile activity of isolated strips of pregnant rat’s myometrium during pregrepresent longitudinally oriented strips; the open bars represent circularly oriented strips. The of the molar concentration of prostanoid producing a half-maximal response. Maximal tension developed in response to the prostanoid; units are N/cm2. Values marked “+“ are significantly of pregnancy; values marked ““ are significantly different from Day 10 of pregnancy.
on rat myometrium
Figs. 2 and the changes
I
10
been previously studied during pregnancy. In all major spects, these effects were analogous to the responses PGF2c. (compare hypothesis that
0
#{149}t#{149}t
20
ufinfi
H
4
u
12
FIG. 5. Effects of nancy. The solid bars pD2 is the negative log is the maximal tension different from Day 22
The
x
on Day change to PGE2
changes circular
in sensitivity muscle
15 than on either in electrophysiological observed
was Day
by Kuriyama
between
Days
significantly 10 or Day sensitivity and
18 and
more 22. Thus of the
Suzuki
22,
sensitive the marked rat’s uterus
[11] was
not
re-
flected in its contractile behavior. No signfficant changes in the number of high-affinity binding sites for POE2 in the rat myometrium were detected during the later part of pregnancy [22]. These results are consistent with our observations, although this consistency should not be overinterpreted. For instance, the EP receptor has been subdivided into three subtypes, the EP1, EP2, and EP3 receptors. Current evidence
suggests
that
both
EP1 and
EP3 receptors
are
pres-
PROSTANOIDS
AND PREGNANT
MYOMETRIUM
399
U-46619 10
A
2 8
0 U)
3.
2 01
w
6
I-
a
S
0. 4
2
S
I
4
2
S
0
I
I
I
10
I
I
12
14
DAY
OF
I
I
I
16
I
20
18
10
22
12
14
DAY
OF
12
14
DAY
OF
PREGNANCY.
10
16
18
20
22
20
22
PREGNANCY
4
2 8
0 U)
01
a
0.
S
0
I
I
I
10
I
I
12
14
DAY
OF
FIG. 6. Effects of nancy. The solid bars pD2 is the negative log is the maximal tension different from Day 22
ent
in rat’s
myometrium
[23].
I
it is possible that three present simultaneously
I
16
I
ii
S 4
S
0
I
18
20
22
10
late-pregnant does
not
However,
under
cyclase with the
different binding in the rat’s uterus.
human
uterus
[16],
appear
to
inhibited
be
some
cir-
in this tissue EP2 site. Thus
sites for PGE2 are These sites would
the
late-pregnant by
rat’s
a mechanism
coupled to the IP receptor. The low potency of iloprost in causing contractions of the myometrium (see Fig. 5) supports the notion that the compound can act as a weak agonist
at other
prostanoid
receptors.
under certain circumstances, rat’s myometrium [24, 25], use
of the
stable
selective
Thus,
although
stimulate adenylate there is no evidence analog
16
18
PREGNANCY
U-46619 on the contractile activity of isolated strips of pregnant rat’s myometrium during pregrepresent longitudinally oriented strips; the open bars represent circularly oriented strips. The of the molar concentration of prostanoid producing a half-maximal response. Maximal tension developed in response to the prostanoid; units are N/cm2. Values marked “+“ are significantly of pregnancy; values marked ““ are significantly different from Day 10 of pregnancy.
probably be regulated independently. Such a possibility may explain the apparent change in affinity of POE2 binding sites in the rat’s uterus that occurs postpartum [22]. Alternatively, PGE2 effects on adenylate cyclase may be mediated through IP receptors. There were no radical changes with pregnancy in the effect of the P012-mimetic compound iloprost. Thus, unlike uterus
:
PREGNANCY
cumstances, PGE2 stimulates adenylate [24], a property normally associated
the
3
2 Ui I-
iloprost
([17];
PCI2 cyclase from this
can, in the
study)
or the
even
more
selective
analog
cicaprost
(Crankshaw
and
Gaspar, unpublished results) that it can directly affect uterine motility in the rat through action at IP receptors. It therefore seems unlikely that the myometrium is the target for the large amounts at the end of pregnancy
of PG!2 [18].
produced
by the
rat
uterus
In a similar fashion, pregnancy did not bring about a radical change in the response of the rat’s uterus to the Txmimetic U-46619 when compared to the nonpregnant state [17].
The
compound
maintained
that its effects were not ceptors. This conclusion
mediated is at odds
a low
potency,
through with the
suggesting
action at TP reobservation that
platelet-generated TxA2 had a potent effect on rat uterine motility [26], although it is possible that those effects were mediated by short-lived product of arachidonate metabolism other than TxA2. They are consistent with the the Tx synthesis inhibitor, OKY-046, reduced TxA2 tion by the pregnant rat’s uterus without affecting
fact that producits mo-
tility [27]. As with PGI2, it is unlikely that the myometrium is the target for the large amounts of TxA2 produced by the rat’s
uterus
at the
end
of pregnancy
[181.
400
CRANKSHAW
The
results
of this
study
suggest,
major question that we posed, duce changes in the receptivity drastic as to indicate that PGI2 ulator of activity during role of these agents must PGF2,,
sults these
did
two
studies
not
pregnancy. be sought.
the
wide
suggested Finally,
Another Although
response
to
to
third
within
the
we
during
trical
13. Tuross
physiologic
control
of the
uterus
Biol
and
in parturition.
14. Anderson
in
4:45-66. 2. Aiken tion.
York: 3. Vane
JW. PG’s and
PG synthetase
In: Robinson
HG.
Raven
Publishers;
JR, Williams
Prostaglandins.
R, Dukes
anti-inflammatory 1972;
of the
of prostaglandin
Br J Pharmac
rat
and func-
Inhibitors.
New
Publishers;
48:629-639.
which
The
of labour
with
prosta-
AL.
Delay
of parturition
in the
of prostaglandin.
rat by Nature
240:37-38.
7. Guthrie
HD.
Control
of time
of parturition
in pigs.
J Reprod
Fertil
suppl
1985;
NH,
16. Dyal
PD, Pashen RL, Jeffcott (fluprostenol) to induce foaling.
9. Liggins sheep.
GC,
Fairclough
In: KnightJ,
Publishers;
J Reprod
RJ, Grieves
O’Connor
1977: 5-25.
LB. The
use of synthetic Fertil
Suppl
prostaglandin 1979;
SA, Forster CS, Knox
M (eds.),
The Fetus
and
Birth.
analogue
27:521-529.
BS. Parturition in the New
York:
and
longitudinal
1981;
Blake
of effects
Reprod
JM.
Effect
rats; comparison
of oxvtocin
and
pros.
the pregnant
from
rat.
of indomethacin
of circular
and
aspirin
mus-
and longitudinal
24:359-372.
BR, Egner
DA, Ghodgaonkar 1982;
of the human
PG. Thromboxane
F2,, production
B2, 6-keto.pros-
by contracting
pregnant
rat uteri
26:281-288.
DJ. The effects
R, Crankshaw
tractility
of the preg-
242:243-250.
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T, Marshall
F,,,, and prostaglandin Biol
1976;
37:348-355. in pregnant
Reprod
in vivo.
lower
uterine
of some
synthetic
segment
in vitro.
prostanoida on the conAm J Obstet Gynecol 1988;
58:281-285.
BC, Burcea
17. Wainman
I, Crankshaw
rat myometrial
DJ. The effects
longitudinal
musde
of prostanoids
in vitro.
Biol
on estrogen39:221-
Reprod
1988;
CA. Poyser
Elsevier
NL. Prostaglandins,
Br J Pharmacol DB.
Multiple
and the pregnant
thromboxanes
rat uterus
1981; 73:75-80. range
and
multiple
F tests.
Biometrics
1955;
11:1-42.
JM. Effects of catecholamines on circular and longitudinal uterine muscle of the rat. EurJ Pharmacol 1981; 76:157-165. 21. Crankshaw DJ, The sensitivity of the longitudinal and circular muscle layers of the rat’s myometrium to oxytocin in m2ro during pregnancy. Can J Physiol Pharmacol 1987; 65:773-777. 22. Moln#{225}rM, Hertelendy F. PGF2,, and PGE2 binding to rat myometrium during gestation, parturition, and postpartum. Am J Physiol 1990; 258 (Endocrinol Metab 21): E740-E747. 23. Coleman RA, Kennedy 1,Humphrey PA, Bunce K, Lumley P. Prostanoids and their El-list,
Marshall
receptors. In: Hansch of Chemical
The
Rational
Compounds,
C, Lammes vol
Evolution
from
PG, TaylorJB
Mechanistic
Design,
3, Membranes
Press; 1990: 643-714. 24. Tanfln Z, Harbon S. Heterologous
regulations a stimulatory
(eds.),
Comprehensive
Study and Therapeutic and Receptors. Oxford: of cAMP
responses
to an inhibitory
prostacyclin effect. Mol Pharmacol 1987; 32:249-257. 25. Vesin M-F, Khac LD, Harbon S. Prostacyclin as an endogenous osine cyclic 3’, 5’-monophosphate levels in rat myometrium
Mol
26. Dubin
33:229-244. 8. Rossdale
1987;
myometrium.
1979; 76:769-782.
the biosynthesis
inhibit
MF (ed),
In: Cuthbert
on the dec. J Physiol 1976;
PGF2,,, on the uterus
Pharmakol
JM. Comparison
M, Marshall
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Chemistry:
1972: 23-124.
Br Commonw
M, 5later SR. Walpole
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of prostaglandins.
Heineman
Gynaecol
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RB., Hillier K, Patel RC. Induction
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6. Chester
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289-302
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Prostaglandin
contributions
uterus
London:
SMM,
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1974:
KI. The
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Vane
of labor.
228.
1980;
Perinatol
rat myometria.
of prostaglandin,
G, Kawarabayashi
Biol
18. Phillips
thromboxanes
Semin
of induction
E2 and oxytocin
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Arch
F2,, on circular
Reprod
at term.
prostacyclin,
and
and
JM. Action
N, Mahtani
taglandin
dominated
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I. Failure
of prostaglandin
H. Effects
Naunyn.Schmiedebergs
taglandin
pregnancy.
GC. Role of prostaglandins,
Suzuki
of hormone.treated
0, Marshall rat.
19. Duncan
MJ, Liggins
on rat pregnancy.
260:335-349. 12. Reiner
20. Chow
Novy
effects
23:410-416.
H and
activity
15. Dubin
major
have no knowledge about changes rates of the different prostanoids
myometrium
11. Kuriyama
cle.
question, the different pattern of responses to PGE2 and PGF2C,, during pregnancy suggest that these prostanoids do indeed act at different sites in the myometrium. This conclusion is supported by binding data [22] but must be tempered by the fact that in the biotransformation
1973;
on uterine
of separate
our
Prostaglandin
Steril
nant
between
by comparison
in
AR
Fertil
our reidentical
differences
GASPAR
10. Fuchs
first
physiological responses
regulated differently, were tested under
reveal
compounds [11, 13].
to the
that pregnancy does not inof the rat’s myometrium so or TxA2 is an important reg-
and PGE2 appear to be in which both prostanoids
circumstances
in answer
AND
Pharmacol
Medicinal Application
Pergammon in pregnant
prostaglandin
rat
E2 and
modulator of adenand endometrium.
1979; 16:823-840.
NH, Blake DA, Egner PG, Ghodgaonkar BR. Effect of platelet-generated thromboxane on contractions of the pregnant rat uterus. Biol Reprod 1982; 26:289295. 27. Ghodgaonkar BR, Dubin NH. Effect of OKY-046, a selective thromboxane inhibitor on prostaglandin production and function of pregnant rat uteri and platelets. Prostaglandins Leukotrienes Med 1987; 26:33-46.
OF
46,
REPRODUCTION
Effects
392-400
(1992)
of Prostanoids
on the
Rat’s
Myometrium
D. J. CRANKSHAW Department
of Obstetrics
and
and Gynecology, Hamilton, Ontario,
In Vitro
during
Pregnancy1
V. GASPAR
Health Sciences Canada L8N
Centre, 3Z5
McMaster
Universiiy
ABSTRACT The
objectives
metrium
PG
of this
vitro
in
(prostaglandin)F,,,
Days
10,
circular
were
study
to a number
the
PGD,,
15,
18, 20,
strips
were
21,
10;
thereafter
potency
than
at Day
10. In contrast,
fell
PGF2,,
Responses value
on Day
for PGE2
were
no changes
there
to PGE2
and
during
PGF,,,
pregnancy
sthps
18 and
then
and
a high
gradually
in potency
the period
response
studied
were
U-46619 but
until
maximal
to
investigated
on
it
(longitudinal
the
that
hypotheses
these
prostanoids
act at different
lower
still
was
or a slight
muscle)
Both ioprost and U-46619 maintained a low potency throughout is probably not a target for PGI2 or TxA2 and that the different support
and
on Day
response
22, when
Day
myo-
longitudinal
between
low
of study
of the Responses
separately.
differences
potency
increased
over
contractile
were
A2-mimetic
prostanoid-dependent, with
on the
rat
(Tx)
thromboxane
patterns,
similar
to a minimum
increase between Days 15 and 21 (circular muscle). We conclude that the pregnant rat’s myometrium responses
were
in the oriented
circularly
the
and
produced
PGD,
of pregnancy and
iloprost,
PGI2-miznetic
and
effects
the
Longitudinally
22 of pregnancy.
and
small.
to determine
of prostanoids.
pregnancy.
patterns
of
sites within
the
myometrium.
INTRODUCTION Prostanoids rition muscle
are
thought
to play
[11, in part through their direct of the uterus [2,31. Several
a major
role
in partu-
effects on the smooth observations have led
analogs and
effectively
induce
labor
the horse [8] but not The role that prostanoids
close
to term
in vitro
suggest
that
in the
it
becomes
unclear. Some on longitudinal more
[7]
membrane an increase on
depolarization. in sensitivity
longitudinal
pared
to Day
muscle 19 pregnant
Accepted
October
18,
Received
August
1, 1991.
needed discharges
to
produce without
rats,
Day
22
although
pregnant the
rats maximum
however,
circular
on Day [10] was
21 than unable
of PGEI, PGE2, were successful.
or aspirin-treated
these agents produced some metrium through mechanisms prostanoid synthesis. There are no reliable data prostanoids
an inmarked
comef-
the maximum muscle from muscle
reDay was
on Day 17. to induce par-
or PGF28,, Furthermore,
although intra-
by prostanoid infusions than they were on Day that the characteristic be brought about by a
interaction between a number of prostanoids. they interpreted the failure of PGF28, to restore electrical activity to muscle strips isolated from in-
domethacin-
as term
Reiner and Marshall [12] found to the mechanical effects of PGF2,,, from
rats;
more sensitive intact rat, Fuchs with infusions of oxytocin
complex However, normal
approaches. Thus Kuriyama and Suzuki [11] investigated electrical but not mechanical responses to PGE2 and found that between Day 15 and parturition there was a marked decrease in the concentration crease in the frequency of burst
21 pregnant
50% of longitudinal
uterine pressure changes induced were less on Day 22 of pregnancy 21. Anderson et al. [14] recognized myometrial changes at term may
invesmus-
sensitive
that produces for PGF28, on
turition infusions
its
pig
concentration sponse) values significantly In the
in the sheep [9] or the rat [10]. play in stimulating uterine ac-
tivity at term in the rat is particularly tigations of the effects of prostanoids cle
[3]. to prostanoids; (PG) F28, or
similar on both days. In contrast, Tuross et no significant differences in EC50 (effective
15, 17, or
to the formulation of this hypothesis: the prostanoids are potent modifiers of myometrial contractility [4]; labor can be induced by prostanoid administration [5]; parturition can be delayed by administration of prostanoid synthesis inhibitors [6]; and prostanoid concentrations in the uterus undergo acute changes around the time of parturition Not all species respond in the same way thus it has been claimed that prostaglandin
fect appeared al. [13] found
during
pregnant
rats
of their other
effects than
concerning
pregnancy,
as evidence
the
although
that
on the inhibition effects
some
myoof
of other
evidence
suggests that they may play an important Dubin et al. [15] have presented evidence rat’s uterus produces substantial amounts
role. For instance, that the pregnant of thromboxane
(Tx) A2, which they claim ity. In the lower segment
myometrial human
could regulate of the pregnant
activuterus,
the stable Tx-mimetic U-44069 has been shown to be a potent uterotonic [16], whereas in the nonpregnant rat’s uterus a similar compound U-46619 is almost ineffective [17]. Similarly, the pregnant rat’s uterus produces large amounts
1991.
‘This work was supported by an operating grant from the MRC of Canada. It was presented in part at the Philippe Laudat Conference on the Biochemistry and Pharmacology of the Myometrium, Alsace, France, September 30-October 4, 1990. tCorrespondence: DJ Crankshaw, McMaster University, Dept of Obstetrics & Gynecology, 1200 Main St. West, Hamilton, Ontario, Canada LEN 3Z5. FAX: (416)5770471.
of PGI2 are potent ment 392
[18];
ineffective
and
despite
on the
the
nonpregnant
fact
that rat’s
stable uterus
PGI2
mimetics
[17], they
inhibitors of pregnant human lower uterine activity [16]. Three major questions stimulated
are segthe
PROSTANOIDS present
studs’.
sitivity metics
First,
are
there
drastic
of the pregnant rat’s uterus that might indicate that the
important ond, when
regulators measured
responses
of the
uterus
to
in the
sen-
to PGI2- and TxA2-minatural compounds are
of contractility under similar rat’s
changes
PREGNANT
AND
during pregnancy? Seccircumstances, are the
PGE2
and
PGF2C, different,
as circumstantial evidence would suggest [11, 13], thus perhaps indicating different roles for the two prostanoids? Finally, can the possible regulation of prostanoid responses during pregnancy give any clues to the question whether different prostanoids To find answers
act to
at different sites [17]? these questions, we examined
the
MYOMETRIUM
out, the tension was allowed justed to 10 mN if necessary), iment
proper
was
Measurement
and
storage
periods. of these
of the
square centimetre Spontaneous
second
half
of
AND
Animals Timed-pregnant River facilities ad
sperm housed
rats
were
St. Constant,
at 22#{176}C on
water
libitum.
a 12L: 12D
The
suppliers
purchased
from
PQ.
They
were
cycle
and
allowed
designated
Charles
kept
the
Strips
in our
food
and
morning
that
were found in the vagina after females had been overnight with a male rat as Day I of pregnancy.
When they
Wistar
Laboratories,
animals
were
normally
allowed
delivered
to reach
on
the
term
morning
in our
facilities,
of Day
in
used
ments,
only
of each used
The
Animals were killed ignated day of pregnancy.
by a blow to the head on the The entire uterus containing
products
was
(95% 02: temperature.
C02) physiological The PSS had the
5%
KC1, 4.6; MgSO4, 1.16; NaHC03, 21.9; glucose, Uteri removing
removed
were the
trimmed fetuses,
and
placed
direction. No attempt and circular muscle
dometrium. taken from tied
with
organ resting was
Most often sites adjacent silk
longitudinal to different and
monitored
through
to MA).
mm
before
that
failed and
a Grass Tissues
suspended
being to respond where
a Grass
polygraph allowed
challenged
with
with possible
vertically
isometric
strips strips
were were
90
mM
initial Force
transducer
KC1. Any to .KC1 was
The
taken
from
each per animals
each
strip. by constructing agent as we have
activity In
was
were
not
dose-response
tested
on
each
strip.
(n) refers to both the of strips since, although
were
taken
from
each
used
experiIn all
number of four strips
animal,
each
was
purpose.
sessed
Anal’’sis significance using
of differences
Student’s
test [19] using p < Values are expressed the
t-test 0.05
between
combined
as the as means
values
with
was
a multiple
as-
range
cut-off point for significance. ± the standard deviation
of
mean.
Materials except
E. Schillinger, Schering Chemical, Ann Arbor, as previously described from
Sigma
chemicals
Chemical were
from
iloprost,
which
was
AG, were obtained MI. They were prepared [17]. Indomethacin Co., BDH,
TABLE 1. Spontaneously developed circularly cut strips of myometrium
St.
Louis,
Toronto,
MO,
a gift from
Dr.
from Cayman and stored was obtained and
all
other
ON.
tension in longitudinally from pregnant rats.
and
in a 10-ml
(Grass Instruments, to equilibrate for
a contraction replaced.
Four in the
PSS at 37#{176}C. An on each strip.
FT-03
7-D were
midhorn. and four
and circular fetuses. The
for
spontaneous
prostanoid
a different
All prostanoids
made to separate lonor to remove the en-
bath containing oxygenated force of 10 mN was exerted
connected Quincy,
carded
thread
was layers
115.5;
tissue; without cut from over
the back of a fetus implanted approximately strips were cut in the longitudinal direction circular gitudinal
at room (mM):
1.16; NaCI, 0.01.
of fat and connective strips of uterus were
desthe
in gassed
salt solution (PSS) following composition
CaC12, 2.5; NaH2PO4, 11.1; indomethacin,
10-mm
22.
Preparation
of conception
strips
were determined curves to each
number number
orientation for
over
was determined by taking Activity was expressed as a
experiments. one
at 2 Hz
activity present during in terms of Newtons
record
to assess
cases, the given animals and the
quantified as we digitalization
[17]. In most experiments there was and nine concentrations of prostanoid, of these experiments equal to those in activity was assessed.
dose-response
Statistical Tissue
first
of prostanoids dose-response
described previously one control period making the length which spontaneous
METHODS
output
of pregnancy in succession.
of the
Effects cumulative MATERIALS
amplifier
(N/cm2). activity of muscle
percentage
the
(readexper-
begun.
The mean contractile periods was expressed
strips of pregnancy.
throughout
to baseline mm the
is
activity of the strips was earlier [17] b simultaneous
effects of PGF78,, PGE2, PGD2, iloprost, and U-46619 on the contractility of both longitudinally and circularly oriented myometrium
to decline and after
of Contractility
The contractile have described
at different stages ten such records
rat
393
KCI was
20 strip dis-
washed
Day 10 15 18 20 21 22
of pregnancy
Longitudi 0.9 1.7 1.2 1.7 1.0 1.1
nal strips ± ± ± ± ± ±
Circula
0.3 0.6 0.5 0.7 0.3 0.3
Tissue strips were set up and spontaneously developed termined as described in Materials and Methods. Values are means ± SD from 10 animals in each case;
0.9 1.0 1.3 1.0 0.9 0.9 tension units
r strips ± ± ± ± ± ±
0.5 0.4 0.4 0.3 0.3 0.5
was
de-
are N/cm2.
394
CRANKSHAW CIRCULAR
AND
LONGITUDINAL
Daytl
GASPAR
tation of the strips. There were no marked differences between days for the same orientation or between tations for the same day.
DaytO
250
In general, mained fairly
150
or of the 50
once constant
orientation
of the
of the experiment. This (right panel, longitudinal DayI5
DaylS
250
Absolute
150
in the two muscle Day 22 of pregnancy
‘LI
50
-J
4 >
-J
nflflli. DayiB
0
values
DayIO
I-
z
0 50 4 U)
strips that
UHfldfl
250
150
50
1
I0 4
noon”.
duud.
Oay2i
Day2l
250
I-
z 0 C)
150
0ay22
90
10
TIME
30
50
70
90
expressed as a percentage of the activity in the n = 10-12, and errors are standard deviations
of the means. The elapsed time between the and of one measurement riod and the beginning of the next was less than 1 mm. Right-panel, gitudinally oriented strips; left-panel, circularly oriented strips.
pelon-
1 shows
veloped
by control
periment
proper,
the
values strips
against
of the during the
spontaneous the
day
on
PGE2,
prostanoid Iloprost
first
developed
was
10 mm
of pregnancy
force
circularly
consistent greater
oriented
observation tension. Re-
small.
activity
de-
of the and
The
sensitivity
maximum tension Days 18 and 20 for in responses
Day
10 and
of Day
de-
20; therethe values
development PGF28, and
to PGE2
subsequently
10. Maximum
making
declined tension
the
general
(Fig.
on
4) was
to values
generated
pattern
less exaggerated. (Fig. 5) was excitatory
TABLE 2. Effects of prostanoids from Day 22 of pregnant rats.
Agonist PGF2 PGD2 PGE2 lloprost U-46619
RESULTS Table
developed
and
close
on Day
ex-
orien-
± ± ± ± ±
0.3 0.5 0.5 0.4 0.4
with
during
in circular The pattern
on longitudinal
strips
10
preg-
strips on of change
Maximal force (N/cm2) 1.6 ± 0.7 3.5 ± 2.7 2.4 ± 1.5 2.7 ± 2.0 3.6 ± 0.2
0.63
2.5 0.5 1.6 16
Negative log of the molar concentration giving ± the standard deviation of the mean. bMicromOlar concentration giving a half-maximal
this
of myometrium
EC (M)’
pD2 6.2 5.6 6.3 5.8 4.8
of change
at all times
nancy, although the tension developed Days 10 and 15 was extremely low.
(mm)
FIG. 1. Spontaneous contractile activity in strips of myometrium isolated from pregnant rats. Activity was measured over 10-mm periods as described in Materials and Methods. The height of each bar represents the activity in that time period first (control) time period;
maximum
longitudinal
of Day 10). In contrast, reached its peak between Day 22 for PGD2. The pattern of change
for 70
duration
by PGE2 was higher in both longitudinal (2-fold and 1.4fold respectively) and circular strips (4.1-fold and 4.5-fold respectively) than that generated by either PGF2C. or PGD2. However, the absolute maximum values were generally lower
Day22
50 50
between
tension
to those
150
30
and
creased, reaching a nadir between Days 18 and after it slowly increased (though it never reached
than
250
10
the
quite different. In longitudinal strips there were no significant changes in sensitivity throughout the study period, whereas in circular strips sensitivity was greater on Day 15
ui
50
for
layers in response to the prostanoids on are shown in Tables 2 and 3, respec-
were minimal except for the longitudinal strips developed
imum
>.
I> I.C.) 4 uJ
strips,
sponses to PGF2Q and PGD2 (Figs. 2 and 3) changed with very similar patterns. In early pregnancy, the rat’s uterus was highly sensitive to these two prostanoids, but the max-
Day2O
Day20
4
muscle
developed it reday of pregnancy
is shown graphically in Figure 1 strips; left panel, circular strips).
for sensitivity
Differences 150
U-
activity of the
tively. Changes in sensitivity and maximum developed forces of longitudinal and circular muscle strips to the prostanoids PGF28,, PGD2, PGE2, iloprost, and U-46619 with advancing pregnancy were complex and are represented in Figures 2-6, respectively.
250
0 0
spontaneous irrespective
either orien-
a half-maximal response.
response
PROSTANOIDS of sensitivity to iloprost resembled major exception: in both longitudinal there and
was
a significant
22. The Tx-mimetic
on
strips
U-46619
of both
be detected nor maximal
drop
(Fig.
between
6) had no
Day 18, changed
PREGNANT
a very
low
significant
whereafter significantly.
Days
could
sensitivity
uterus factors:
to prostanoids the uterus
develops
spontaneous
processes
are
[15]. In our performing
are itself
complex,
since
hampered produces
by at least prostanoids, activity.
by the the in the has been tissues
of the
duration
two and
major also
it
of
EC 6.0 ± 0.4 5.3 ± 0.2 6.2 ± 0.6 5.6 ± 0.5
1.0 5.0
U-46619
4.9
spontaneous
activity
of the
log
the standard
molar
1.3 ± 0.3 2.2 ± 1.2 1.1 ± 0.7 1.3 ± 1.2 1.3 ± 0.3
2.5 12.6 concentration
deviation
of the
concentration
giving
pregnancy by
prostanoid problem
in the
sponse
experiments
giving
a half-maximal
a half-maximal
response.
of the
difficult,
is
presence
1 are
since
spontaneous
activity
different
of tissues
obtained
shown
on
those
reported
from
Table
by others.
period measured in a stepwise
0
z
2
22 DAY
OF
PREGNANCY
OF
16 18 PREGNANCY
4
z
0
3
a, 2
Ui
I-
01
a
2
x
0 14 DAY
FIG. 2. Effects of nancy. The solid bars pD2 is the negative log is the maximal tension different from Day 22
OF
16 18 PREGNANCY
20
22
12
14 DAY
in this
1 and
Figure
Dubin
et al. found dur-
after a 15-mm equilibration fashion from Day 15 to Day
O IJJ I-
the
used
oriented strips only and activity, i.e. activity occurring
z a
presents
baseline against which an accurate description
profile
[15] studied longitudinally that the initial contractile ing a 15-mm time, increased
essentially
it
PGF2
12
response
mean.
with a constantly moving responses. Therefore,
study was necessary. The results we
of mndomethacin has been reported to be a time-dependent phenomenon [15]. Changing spontaneous activity over time within a given tissue makes the interpretation of dose-re-
10
force
(N/cm2)
0.63
± 0.1
from
Maximum
PGF2 PGD2 PGE2 lloprost
observers to measure
these
first problem was treated presence of a concentration shown to inhibit [15]. The second
of myometrium
pregnant
Both of the
strips
(p.M)b
bMicromolar
responsiveness
contractile
influenced experiments, all studies
of mndomethacin that production in similar more
the
on circular
pD2
‘Negative
DISCUSSION to describe
of prostanoids rats.
Agonist
±
Attempts
395
21
potency
effects
neither
MYOMETRIUM
TABLE 3. Effects Day 22 of pregnant
of PGE2 with one and circular strips,
in sensitivity
orientations:
before tension
that
AND
20
PGF2 on the contractile activity of isolated strips of pregnant rat’s myometrium during pregrepresent longitudinally oriented strips; the open bars represent circularly oriented strips. The of the molar concentration of prostanoid producing a half-maximal response. Maximal tension developed in response to the prostanoid; units are N/cm2. Values marked “+“ are significantly of pregnancy, Values marked “*“ are significantly different from Day 10 of pregnancy.
396
CRANKSHAW
AND
GASPAR
PGD2 4
10
z
8
0 U)
3
z
w I-
2
fHii
:
4
1
0 10
12
14
DAY
OF
16
18
20
22
10
PREGNANCY
12
14
DAY
OF
16
18
20
22
PREGNANCY
4 2 0 C’, 2
w
*
I-
01
a
0. *
4
0 10
FIG. 3. nancy. The
12
14
DAY
OF
16
18
20
22
n
I-I
10
PREGNANCY
Effects of PGD2 on the contractile solid bars represent longitudinally
activity oriented
Fi
t t
of isolated strips strips; the open
12
14
DAY
OF
16
18
20
22
PREGNANCY
of pregnant rat’s myometrium during pregbars represent circularly oriented strips. The
P02 is the negative log of the molar concentration of prostanoid producing a half-maximal response. Maximal tension is the maximal tension developed in response to the prostanoid; units are N/cm2. Values marked “+“ are significantly different from Day 22 of pregnancy; values marked ““ are significantly different from Day 10 of pregnancy.
22 of pregnancy.
In our
ferences in spontaneous 10-mm interval, which was suspended in the observed
a steady
decline
case,
there
were
no significant
dif-
activities measured during the first began about 45 mm after the tissue bath. Furthermore, Dubin et al. [15] in spontaneous
activity
over
a 60-
therefore surprising to find that in response to the prostanoids there were not any significant differences between the two layers. Tuross et al. [13] stated that “it is preferable to isolate the two muscle layers as completely as possible,
21 for lonWe cannot
since the layers are electrically coupled and the contractions of one layer are synchronized with those of the other, especially at term.” We chose not to separate the layers; our rationale for this was that the interaction between the layers is probably of physiological significance and that our in-
explain the difference between the two studies since, except for the strain of rats used (Sprague-Dawley versus Wistar) the conditions seem to be identical. Nevertheless, these
terest was in how the different layers behave when they are coupled. Furthermore, what we believe to be important physiological differences between the two layers can be
results do indicate that our method of assessing responsiveness was not compromised by fluctuations in baseline. There are a number of circumstances under which longitudinal and circular muscle of the rat’s myometrium
demonstrated when contact between them is unperturbed [21]. Similar logic prompted our decision to leave the dometrium intact. The pattern of responsiveness of the rat’s myometrium
behave quite differently. For instance, the relationship between mechanical activity and the underlying electrophysiological events is markedly different between the two layers [14]: during pregnancy, the two layers react differ-
during pregnancy pare Figs. 2-6). seen, we Tuross
ently
tO
mm period for most days of pregnancy, whereas our observations showed activity to remain relatively constant. There was a greater variability between tissues gitudinal strips and on Day 22 for circular
to catecholammnes
[20]
and
on Day strips.
to oxytocin
[13, 21]. It was
PGF2a
is clearly prostanoid-dependent Because of the complexities of the
will discuss et al. [13] on
Days
each studied 15,
prostanoid in vitro
17, and
21
in turn. myometrial in a colony
en-
(comchanges responses
of rats
that
PROSTANOIDS
AND PREGNANT
MYOMETRIUM
397
PGE2
2
0 U) 2 Ui
01
I-.
a a
4
10
12
14 DAY
16 18 PREGNANCY
OF
;
20
22
t
1:
4
2
6
3.
0 a, 2
5
Ui
01
I-
0
4
a
2
3 4
2
1#{149}
H
1
.1
-
0-
10
12
14
DAY FIG. 4. Effects of nancy. The solid bars pD2 is the negative log is the maximal tension different from Day 22
normally and
delivered
some
ours.
on
notable
They
found
that
ogous
the
muscle between
to those
Days
(0.63
and
pM).
12
14
DAY
16
OF
some
findings
to PGF2U
15 and
21; these
was
right
and no anal-
panel).
Sim-
0.83 j.&.M in longitudinal and are close to those we obtained
responses
to PGF2a changes
(compare they tween
between upper
found Days
to be
on
Day
21
fraction
and
15 and
[13]
circular
muscle
The
lower 21.
found only
magnitude
longitudinal
a progressive
a change within the
number of receptors in coupling between
right
on
Days
of responses and panels
increase It is possible
in
circular of Fig.
21.
to PGF2a muscle
circular that
17 and
strips
muscle the
differences
time. nancy on
2), whereas be-
role
nomena
in the target
Day
any of these
22 (Fig. throughout
panels).
the
later
sites
whole
over change.
differ-
with
this
for PGF2U
rat myometrium time
Day was
15.
Al-
complex,
response of the myobe regulated; among
biotransformation rate tissue; (2) a change
processes
binding
the
a significant
binding
from
is almost
2, left
between
demonstrated
in the target the receptor
observed
by
used.
at which the in vitro might
The increase in PGF2a [22] is consistent with
correlation sponse
that we
explained
compared
of changes significant
always
be
[22]
of pregnancy
though the pattern this was the only
differences. were
graded
22
of high-affinity
membrane
significant to prostanoids
et al.
in the
in
late
number
in this case both In our study,
We
a reduction
in a crude
can
Hertelendy
in the
(1)
Tuross
20
preparations
these are prostanoid
whereas
found
increase
studies
muscle
are many sites to prostanoids
to be similar
et al. [13]
two
in the
Moln#{225}rand
was are
the
ences
greatest
there results
2 (upper
between
similarities
their
17, although
layers showed the responses
18
PREGNANCY
There metrium
found
Tuross
10
22
sensitivity of both muscle layers between Days 17 and 21, but the changes were significant only in the circular layer. We made similar observations between Days 18 and 22, but
graded,
1.0
are
Day
and
20
PGE2 on the contractile activity of isolated strips of pregnant rats myometrium during pregrepresent longitudinally oriented strips; the open bars represent circularly oriented strips. The of the molar concentration of prostanoid producing a half-maximal response. Maximal tension developed in response to the prostanoid; units are N/cm2. Values marked “+“ are significantly of pregnancy; values marked ““ are significantly different from Day 10 of pregnancy.
between
in Figure
ilarly, EC50 values of 0.36 circular muscle respectively
18
PREGNANCY
response
on
shown
OF
21. There
differences
in longitudinal difference
Day
16
of the in the
tissue; and (3) a change and the final response. might
completely
play
in the
unknown
pheat this
binding sites at term of pregthe increase in sensitivity seen However, site part
number
there and
of pregnancy.
is no
direct
in vitro
re-
CRANKSHAW
398
AND GASPAR
ILOPROST 4
2
3
0 In 2
a
Ui
01
I2
a
4
0 10
12
14
DAY
16
OF
18
20
22
PREGNANCY
8
4
7 -
-
2
6
0
-
3
a) 5 01
2
__
3
0
‘t
10
p
effects
of PGD2
T
14 DAY OF
r
7
r
%
16 18 PREGNANCY
22
not reto
in vitro
3). On the basis we observed
changes in receptors or post-receptor to changes in biotransformation)
this
of the tenuous are related to
mechanisms suggests
that
(i.e., not PGF2U
and PGD2 act at the same site in the rat’s myomethum. There is now compelling evidence to suggest that the major prostanoids of the 2-series PGD2, PGE2, PGF2,, PGI2, and TxA2
respectively.
the primary that have There
ligand at one been designated is a crossover
tanoid and receptor with a lower potency there
is a wide
of a series of prostanoid DP, EP, FP, IP, and of activity
in distribution
ceptors within different tissues (for view see Coleman et a!. [23]). Evidence tention pregnant
between
(e.g., PGE2 can act at the than PGD2, the primary
diversity
of the
contraction agonists are
TP
pros-
various
re-
a comprehensive supporting our
recon-
of smooth ineffective
site the
smooth sults). ceptors occurring
OF
I
I
I
16 18 PREGNANCY
20
in the DP re-
muscles [23]. on rat uterine
muscle (Crankshaw These points argue in the
rat
and against
22
myometrium.
prostanoids
Gaspar, unpublished the presence of DP Moreover,
tested,
PGD2
only to PGF2a in systems containing ceptor [23]; in these systems, PGD2 of PGF2a. The nature of the different from those
rere-
of all naturally
is second
in
potency
exclusively acts as a less
the FP repotent an-
changes in response to POE2 to PGF2a and PGD2. There
are clearly were no
alog
significant although
DP receptor ligand), and
that PGF2a and PGD2 act at the same rat’s uterus is as follows. Action at
ceptor tends to inhibit Selective DP receptor
14 DAY
have
are each receptors
12
iloprost on the contractile activity of isolated strips of pregnant rat’s myometrium during pregrepresent longitudinally oriented strips; the open bars represent circularly oriented strips. The of the molar concentration of prostanoid producing a half-maximal response. Maximal tension developed in response to the prostanoid; units are N/cm2. Values marked “+“ are significantly of pregnancy; values marked ““ are significantly different from Day 10 of pregnancy.
on rat myometrium
Figs. 2 and the changes
I
10
been previously studied during pregnancy. In all major spects, these effects were analogous to the responses PGF2c. (compare hypothesis that
0
#{149}t#{149}t
20
ufinfi
H
4
u
12
FIG. 5. Effects of nancy. The solid bars pD2 is the negative log is the maximal tension different from Day 22
The
x
on Day change to PGE2
changes circular
in sensitivity muscle
15 than on either in electrophysiological observed
was Day
by Kuriyama
between
Days
significantly 10 or Day sensitivity and
18 and
more 22. Thus of the
Suzuki
22,
sensitive the marked rat’s uterus
[11] was
not
re-
flected in its contractile behavior. No signfficant changes in the number of high-affinity binding sites for POE2 in the rat myometrium were detected during the later part of pregnancy [22]. These results are consistent with our observations, although this consistency should not be overinterpreted. For instance, the EP receptor has been subdivided into three subtypes, the EP1, EP2, and EP3 receptors. Current evidence
suggests
that
both
EP1 and
EP3 receptors
are
pres-
PROSTANOIDS
AND PREGNANT
MYOMETRIUM
399
U-46619 10
A
2 8
0 U)
3.
2 01
w
6
I-
a
S
0. 4
2
S
I
4
2
S
0
I
I
I
10
I
I
12
14
DAY
OF
I
I
I
16
I
20
18
10
22
12
14
DAY
OF
12
14
DAY
OF
PREGNANCY.
10
16
18
20
22
20
22
PREGNANCY
4
2 8
0 U)
01
a
0.
S
0
I
I
I
10
I
I
12
14
DAY
OF
FIG. 6. Effects of nancy. The solid bars pD2 is the negative log is the maximal tension different from Day 22
ent
in rat’s
myometrium
[23].
I
it is possible that three present simultaneously
I
16
I
ii
S 4
S
0
I
18
20
22
10
late-pregnant does
not
However,
under
cyclase with the
different binding in the rat’s uterus.
human
uterus
[16],
appear
to
inhibited
be
some
cir-
in this tissue EP2 site. Thus
sites for PGE2 are These sites would
the
late-pregnant by
rat’s
a mechanism
coupled to the IP receptor. The low potency of iloprost in causing contractions of the myometrium (see Fig. 5) supports the notion that the compound can act as a weak agonist
at other
prostanoid
receptors.
under certain circumstances, rat’s myometrium [24, 25], use
of the
stable
selective
Thus,
although
stimulate adenylate there is no evidence analog
16
18
PREGNANCY
U-46619 on the contractile activity of isolated strips of pregnant rat’s myometrium during pregrepresent longitudinally oriented strips; the open bars represent circularly oriented strips. The of the molar concentration of prostanoid producing a half-maximal response. Maximal tension developed in response to the prostanoid; units are N/cm2. Values marked “+“ are significantly of pregnancy; values marked ““ are significantly different from Day 10 of pregnancy.
probably be regulated independently. Such a possibility may explain the apparent change in affinity of POE2 binding sites in the rat’s uterus that occurs postpartum [22]. Alternatively, PGE2 effects on adenylate cyclase may be mediated through IP receptors. There were no radical changes with pregnancy in the effect of the P012-mimetic compound iloprost. Thus, unlike uterus
:
PREGNANCY
cumstances, PGE2 stimulates adenylate [24], a property normally associated
the
3
2 Ui I-
iloprost
([17];
PCI2 cyclase from this
can, in the
study)
or the
even
more
selective
analog
cicaprost
(Crankshaw
and
Gaspar, unpublished results) that it can directly affect uterine motility in the rat through action at IP receptors. It therefore seems unlikely that the myometrium is the target for the large amounts at the end of pregnancy
of PG!2 [18].
produced
by the
rat
uterus
In a similar fashion, pregnancy did not bring about a radical change in the response of the rat’s uterus to the Txmimetic U-46619 when compared to the nonpregnant state [17].
The
compound
maintained
that its effects were not ceptors. This conclusion
mediated is at odds
a low
potency,
through with the
suggesting
action at TP reobservation that
platelet-generated TxA2 had a potent effect on rat uterine motility [26], although it is possible that those effects were mediated by short-lived product of arachidonate metabolism other than TxA2. They are consistent with the the Tx synthesis inhibitor, OKY-046, reduced TxA2 tion by the pregnant rat’s uterus without affecting
fact that producits mo-
tility [27]. As with PGI2, it is unlikely that the myometrium is the target for the large amounts of TxA2 produced by the rat’s
uterus
at the
end
of pregnancy
[181.
400
CRANKSHAW
The
results
of this
study
suggest,
major question that we posed, duce changes in the receptivity drastic as to indicate that PGI2 ulator of activity during role of these agents must PGF2,,
sults these
did
two
studies
not
pregnancy. be sought.
the
wide
suggested Finally,
Another Although
response
to
to
third
within
the
we
during
trical
13. Tuross
physiologic
control
of the
uterus
Biol
and
in parturition.
14. Anderson
in
4:45-66. 2. Aiken tion.
York: 3. Vane
JW. PG’s and
PG synthetase
In: Robinson
HG.
Raven
Publishers;
JR, Williams
Prostaglandins.
R, Dukes
anti-inflammatory 1972;
of the
of prostaglandin
Br J Pharmac
rat
and func-
Inhibitors.
New
Publishers;
48:629-639.
which
The
of labour
with
prosta-
AL.
Delay
of parturition
in the
of prostaglandin.
rat by Nature
240:37-38.
7. Guthrie
HD.
Control
of time
of parturition
in pigs.
J Reprod
Fertil
suppl
1985;
NH,
16. Dyal
PD, Pashen RL, Jeffcott (fluprostenol) to induce foaling.
9. Liggins sheep.
GC,
Fairclough
In: KnightJ,
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