36, 36 1-368. (1987). 361. Role of 4-Hydroxylated. Estradiol in. Reducing ..... sulfate'. 7 H2 0,. 1.2; glucose,. 11.1). At the laboratory, both arterial segments were.
OF
BIOLOGY
36, 36 1-368
REPRODUCTION
(1987)
Role of 4-Hydroxylated Estradiol in Reducing Ca2 + Uptake of Uterine Smooth Muscle Cells through Potential-Sensitive Channels’ S. L. STICE,3
J. P. ROSAZZA,5
S. P. FORD,2’3 Department
of Animal
Iowa
D. E. VAN
ORDEN4
Science3
University
State
Ames,
and
Arterial
Iowa
50011
and of Obstetrics and Gynecology4
Departments and
Medical
Chemistry5
University Iowa City,
of Iowa Iowa 52240
ABSTRACT Entry of ionic Ca2 + into two major membrane channels. by membrane depolarization, csl-receptor-ligand interactions.
lations
of
suring
the
specific
Ca2
+
Ca2
inhibitors
+
used
through
PSCs,
a
4-hydoxylated
the
blockade +
of
and
Ca2
by agonists
no may
by known
1969).
This
effect
indicate
on ROCs. a refined
lag time, inhibited These
regardless of site by cycloheximide data
effects Recent
suggest
through evidence
Accepted ‘iournal Economics
Paper No. Experiment
and
and
2444; 2
11
that local from
begins of
after
a 20-
administration, (Killam et
estrogen
and is 1973).
a!.,
exerts
its
production of our laboratory
30-mm
a
vascular
mediator. and others
supported
J-11946 Station, in
Kildee
Hall,
request: Iowa
Dr. State
a,nrinone
mechanisms
of the Ames, party
by
Iowa Iowa;
Agriculture Projects
United
States
and 1994, Public
S. P. Ford, University,
Department Ames, IA
of
Animal
was
uterine
The
presence
system
for
choosing
of separate
an
muscle
Ca2
+
appropriate
excluding
the
inter-
was also determined
Ca2 uterine
as their
segments induced by Furthermore, it was
additive, it. It flow
by mea-
as well
two smooth of extracellular
arterial inhibited.
blood
controlling
studies,
By
for opening
increase
This was studied
towards uptake
ROCs.
of uterine be selectively
in pigs, +
decreased
pathways
blood
that Ca2
that
can
+
be
flow.
Gelbke porcine artery
et al., 1975) and are synthesized by the conceptus (Mondschein et al., 1985), uterine (Van Orden et al., 1983a), and endometrium
(S.
Ford,
P.
Orden et hyperemia Home
Iowa
State
al., 1983b), is unaffected
tion of cycloheximidt been reported that bound receptors for target cells (Schaeffer
2443 Health
Grant.
Reprint
sensitivities inhibits
through
and
pigs.
perfusion
University,
and
D.
E. Van
Orden, University of Iowa, unpublished observations). In addition, 4-hydroxylated estradiol (40H-E2) is equipotent with estradio!-17a (E2) in increasing UBF when injected into the uterine artery of gilts (Van
September 4, 1986. May 5, 1986.
Received
Service
in UBF
uptake
+
in
suggests that catechol estrogens (2- and 4-hydroxylated estrogens) may serve as this mediator. These metabolites of estrogen are found in the circulation when estrogens are high (Fishman and Dixon, 1967;
of estrogen results in an increase flow (UBF) in the pig, as has been mammalian species (Dickson et a!.,
increase
arteries in in vitro
different specifically
to
INTRODUCTION
The administration in uterine blood observed in other
uterine arteries
D-600
two separate
a compound
exhibited
Ca2
uptake
+
the
uptake and contractions (ROC activator) could
+
with
of
markedly D-600
inhibits Ca2
pathway
(40H-E2),
PSCs
cells
of uterine
showed amrinone.
specifically
or amrinone, and phenylephrine
Ca2
independently
muscle properties
parameters D-600 and
amrinone
common the
in smooth contractile
These study:
estradiol
through
activated
uptakes. in this
that of
uptake
and
of D-600 activator)
demonstrated
pretation
tone
while
concentration high-K (PSC
vascular smooth muscle cell for contraction is thought to be mediated by The first are designated as potential-sensitive channels (PSCs), which are opened and the second, as receptor-operated channels (ROCs), which are activated by This study was designed to determine the presence of these 2 distinct popu-
channels
entry
baseline
the
Science,
50011.
361
and unlike E2, by simultaneous
its uterine administra-
(Ford et al., 1986). It has also there are specific membranecatecho! estrogen on estrogen et a!., 1980). Catechol estrogens
STICE
362
may The
reach the pig uterus
uterine possesses
artery via a system
is capable of carrying lumen to the adventitial (Magness
and
estrogens surface
Ford,
1982).
estrogens to the uterine rather than a vascular methoxylation
of
0-methyltransferase, (Ball and Several estrogens
is found
a!.
(1978) Ca2
+
marked increase in UBF no effects on the general researchers observed that estrogen
hyperemia
UBF in Redman
response (1984)
observed channel
mo;
13)
cells
further Since time
a a
increase
in
Walters and lag between
the administration of nifedipine and a reduction in blood pressure of pregnant women, we hypothesized a role of hydroxylated estrogens in reducing Ca2 + uptake by smooth muscle cells of the uterine arteries. It is known that uptake of extracellular Ca2 + is required
for
that drugs
vascular that
vascular
Further, membrane an exposure time contractions 30-mm time vasodilation subsequent Membrane
muscle occurs channels
contractions,
and
after exposure to (Bolton, 1979).
Ca2 + channel blockade requires of several mm to block Ca2 k-induced
(Meisheri et a!., lag associated
1981). with
Thus, the 20- to estrogen-induced
may result from its hydroxylation blockade of Ca2 + channels. channels mediating Ca2 + entry
been designated channels (PSCs) cell membrane concentration,
smooth
relaxation block these
as two major are activated
and have
types. Potential-sensitive by depolarization of the
after an elevation of extracellular K and Ca2 + influx through these channels
is selectively Meisheri et
inhibited a!., 1981).
by D-600 (Bolton, Receptor-operated
(ROCs) are phrine-induced
opened by activation
norepinephrineof al-adrenergic
or
1979; channels pheny!ereceptors,
and Ca2 + influx through these channels is selectively blocked by amrinone (Meisheri et a!., 1981). This study was therefore conducted to verify the presence of each type of calcium channel (ROC and PSC) in uterine possible interaction
arterial with
smooth 4OH-E2
muscle
and
gilts of similar age and weight (10-12 kg) that were exhibiting consecutive
the was
estrous
their
At artery
cycle.
designated
Contraction
at doses that had Further, these uteri were under
no
METHODS
cycles of normal duration to be killed during the
of
estrus
that hydroxylated of Ca2 + channels.
to nifedipine. observed a 20-mm
Yorkshire 140-160
estrous assigned
catecholblood
AND
General
that nifedipine, blocker, caused
in the rat circulation. rats whose
exhibited
uterine artery pathway the rapid
by in red
MATERIALS
catechol
via a lymphatic would avoid
Knuppen, 1980). lines of evidence suggest act through blockage
Sandahl et potential-sensitive
of
compounds
which
route. that
from the of the uterine Delivery
artery route those
a lymphatic of lymphatics
ET AL.
The
as Day
(18-22 days) were lutea! phase (LP; Day first
day
of behavioral
0.
Studies
death, a 3.5-cm supplying one
segment uterine
of the middle horn of each
uterine gilt was
excised immediately the mesometrium.
in front of its first bifurcation in Each arterial segment was placed
immediately oxygen,
a container dioxide)
into carbon
5%
(22#{176} C; composition
of oxygenated Krebs-Ringer
in millimoles
per
(95% solution
liter
=
sodium
chloride, 118.1; sodium bicarbonate, 25.0; potassium chloride, 4.7; calcium chloride’ 6 H2 0, 2.5; potassium dihydrophosphate, 1.2; magnesium sulfate’ 7 H2 0, 1.2; glucose, 11.1). At the laboratory, both arterial segments described arteries
were prepared for simultaneous perfusion as previously for ovine and bovine uterine (Ford et a!., 1976). Briefly, arterial segments
were
cannulated
tubing within
and 60
perfused continuously
at
mounted after
mm
each
end
with
polyethylene
in duplicate perfusion chambers death. Arterial segments were
intraluminally oxygenated
and
extraluminally Krebs-Ringer
with solution
(37#{176}C). An extra!uminal and intraluminal perfusion rate of 10 ml/min was maintained throughout the perfusion of each artery. Uterine arterial segments were allowed a 30-mm equilibration period, by which time all arteries had established a constant baseline perfusion flow and
pressure (BPP) against the intra-luminal were ready for the initiation of drug per-
fusions.
Drug
centrations Changes resistance
in the intra-luminal perfusion flow. in perfusion pressure arising from changes in to flow through the arterial segments were
measured recorded Hewlett Ca2
+
dosages
are
with Statham in millimeters Packard Uptake
Calcium previously
7700
reported
as the
pressure of mercury chart
final
transducers (mmHg)
con-
and by a
recorder.
Studies influx was measured reported by Meisheri
by using a technique et al. (1981). Mea-
ROLE surement
of unidirectional
study uptake
phenylephrine (influx). For
exposed
to (10
tissue
during
mm).
primarily
approach
amount
to
Ca2
+
us
to
from the of uterine
+
section
used
for Ca2 + placed into
for
entering
can
This
for
contraction
shown
from vascular 1981). Each
time
weighed,
the
on the
extracellular space. artery immediately
uptake
Brmefly, adjacent
studies
influx determinations. a container of oxygenated
been
to be
determine
or high-K
was
the to
utilized
Segments (95%
363
HYPEREMIA
Ca2 + Lynch,
to
experimental
selectively
UTERINE
Ca24 were the
be assumed
influx.
IN
out
a short
of
periods
of phenylephrine
of Ca2 segment
carried
solutions
short
allowed
influence
the
The
such
were
and high-K4-induced this, the arterial segments
Ca-containing
period due
fluxes
OF 4OH-E2
were 02, 5%
C02) physiological salt solution (PSS) at 22#{176} C, composition in millimoles per liter = sodium chloride, 140; potassium chloride, 4.5; D-glucose, 10; HEPES,
to
and
containing
inhibit
both
placed
0.5
efflux
smooth segment in
ml
of
a separate
Protoso!
PPO PA,
and and
0.3 250
International
g POPOP, ml Triton
Fisher X-100,
Corporation,
Mt.
dissolved in each liter of Fisher Scientific) was added for
radioactivity.
artery,
Additionally,
one
determine
0.5-cm
segments
binding
were
of
were
remaining
attached length
a 12 X 75 nonradioactive
was
culture PSS.
to clamp this length tube, thus suspending in the buffer solution. to
apply
muscle, These the
a constant
to
a 2.4-gram
used
to lower
After
each
tube containing A polyethylene
while
segment
4 ml stopper
of
the into
aerated was used
of silk against the side of the the arterial segment and weight The 2.4-gram weight was used tension
on
and resulted in a more by triplicate 0.5-cm segments tubes were kept aerated and
experiment.
weight,
a 60-mm
the
arterial
smooth
uniform uptake of from each animal. at 37#{176}C throughout equilibration
period,
the rings were exposed to control or experimental solutions containing E2, 4OH-E2, D-600, or amrinone for periods of 30 mm, followed by a 10-mm exposure to high-K4, pheny!ephrine, plus the inhibitor and/or protocol was designed to
At the end of the 10-mm were transferred to tubes Ca2 4-free 10 M maintained
D-600, additions
or amrinone of high-K4
incubation containing
buffer (PSS with CaCI2 omitted) lanthanum chloride (LaCl3) at 4#{176}C. At this concentration,
period, tissues 10 ml of a containing for 60 mm LaC13 has
the
of
was
the
of
segments
(n
relatively
consistent
for
all
uterine
was
used the
to
tissue.
of aerated
M LaC13 for 30 exposure to 0.5 of phenylephrine incubation
period,
ml of a Ca2 4-free at 4#{176}C for 60 mm,
determined
coefficient
multiple
each
in 4 ml
placed in 10 10 M LaC13
uptake
by running
end
were
Scintanalyzed, vial and counted
as
previously
variation, =
calculated
6) of a lutea!
uterine artery in a single assay, averaged an uptake of 237.6 ± 5.7 j.zmoles Ca/kg weight). Nonspecific binding of Ca, arterial
phase
6.9%, tissue which
segments
with (wet was in this
study, averaged 38.8 ± 2.6 i.imoles/kg tissue and may have resulted from binding to nonmuscular components of the arterial wall. Nonspecific binding, which averaged 18.1 ± 1.2% of specific uptake, was subtracted
from specific
Drugs
of and
At
described. The intraassay
obtain
and 0.5 .tCi/ml of Ca steroid. This experimental test the criteria of selective
inhibition by E2, 40H-E2, Ca2 + uptake induced by the phenylephrine.
high-K4.
IL,
Ca to
maintained
Ca
of silk
and
Pittsburgh, Products
segment
and
Two 20-cm lengths of 3-0 silk were passed through the lumen of each segment. Both ends of one length
Pro-
for
arterial
nonspecific
These
Toluene, to each
were again containing
determinations.
Research
Prospect,
tissues buffer
influx
vial
Scientific Research
0.5-cm
Ca24
of and dry,
scintillation
(NEN
Ca2 4-free buffer containing iO mm before and during the 10-mm j.zCi/ml of ‘ Ca in the presence
for
uptake
ducts, Boston, MA) and heated at 60#{176}Cfor 2 h to facilitate the breakdown of tissue. Ten milliliters of a Toluene Triton Scintillation cocktail (7.0 g
[N -2- hydroxyethylpiperazine - N’ -2 - ethanesulfonic acid], 5; calcium chloride, 1.5; magnesium chloride, 1.0). At the laboratory, the excess connective tissue was trimmed away, and each artery was cut into segments
and
muscle cells (Deth was then blotted
and
total
hydrochloride
into and
The 40H-E2 E2 by direct the phenolic purification
tography 1985).
each
artery
to
(Sigma,
St.
Louis,
(A. G. Knoll Pharmaceutical Laboratories, W. Germany), amrinone (Sterling
Winthrop, Rensselaer, New England Nuclear, study. from
for
Chemicals
Phenylephrine MO), D-600 Ludwigshafen,
‘ Ca influx
influx.
on
NY), and 45Ca (12.0 mCi/mg; Boston, MA) were used in this
used in the study was introduction of molecular
obtained oxygen
precursor by Aspergillus high performance liquid
alliaceus chroma-
as previously
described
(Williamson
et a!.,
STICE
364
Statistical For in uterine
perfusion
arterial
by split-plot design with
AL.
response
Analysis in vitro
ET
response
analysis perfusion
data,
treatment
differences
were
statistically
for a completely periods as the
ferences analyzed regression tionships
in Ca uptake of arteries by factorial design (Kirk, analyses were used to between inhibition of
uterine treatments.
arterial
segments
‘
evaluated randomized subplot; dif-
from gilts were 1968). Simple determine relaCa uptake of
subjected
to
different
perfusion
sequence
In
Vitro
new
Experiment evaluate artermal
la.
The
the role responsiveness
segments perfusions.
from On
first
of
study
was
4OH-E2 in in vitro.
5 LP gilts were each experimental
period,
arteries received intra!uminal flow, 200
mM
luminal pressures
for
BPPs a bolus which
5 sec;
were
uterine arterial
to both
recorded
and
increase Ten mm a bolus
BPP,
the
vessel).
infusion,
mm
of
4OH-E2
the to
and phenylephrine activity of PSCs muscle membrane.
40H-E2 studies
used was to decrease
and
arteries (raninfused with vessel), and the 30 mm (control during
was used to estimate ROCs on the vascular The concentration of
which
using
studies
to
be
in the
middle
of their
doses from
with (p