BIOLOGY
OF
REPRODUCTION
35,
100-105
(1986)
Adenylate Cyclase in the Rat: to a Decidual-Inducing Stimulus’
Uterine
Responses
R. B. SANDERS,2 F. S. ABULABAN,4 Departments
A. M. BEKAIRI,3 and J. M. YOCHIM5
of Biochemistry2
and
University Lawrence,
Physiology
and
Cell
Biology5
of Kansas Kansas 66045
ABSTRACT
The properties inducing
sage
of uterine
stimulus
to the
uterus,
no
uterus,
adenylate 4 of
on Day
changes
(2.73 vs. 2.74), velocity (Vmax) broadened from
nineteen
was
0.3 an
mM
fold before
activation
from
in resistance
additional
5.8 mM
and
of
to
AC
external
by
Michaelis
by
cyclic
adenosine
stimulus Thus major
several factor
plantation Previous adenylate
Accepted December 26, 1985. Received September 19, 1985. ‘This work was supported in part Biomed Grants 4810, 4171 and 4236. 2 Reprint requests.
College
of Pharmacy, of
Science,
of
ion.
for
The
application
of a decidual-
application
After
adenosine
inhibition
of a gentle
the trauma
masto the (A TP,
5’-triphosphate
that
concentration
(IC50)
a decidual-inducing
microenvironment
this rats
at
of
cAMP and
is a im-
the
for
which
in-
CaCl2
stimulus
cyclase,
has shown that which catalyzes
NIH
Grant
The
University
The
University
HD11797
and
terone
(Bekairi
Bekairi
et
the was
resulted protected
it
and
Saud,
of
Saud,
day of maximal the pattern and
sensitivity magnitude
to of
were replicated in ovariectomized daily injections of estrone and progeset
al.,
al.,
1984a).
1982; These
Sanders
et
studies,
in the uterus and were regulated
To understand the mechanism present study was undertaken. made of selected biochemical
al.,
which the
1983; revealed
sensitivity similarly
to by
the concept that role in transducing of this phenomenon, An examination properties of
AC
after a decidual inducing stimulus was applied to the uteri of rats during Day 4 of progestation. Radiochemical analyses of AC activity were made in uterine homogenates, and the following parameters were examined: apparent Michaelis constant (Km) for adenosine (Vmax),
KU
of King King
phenomenon receiving
the
ovarian steroid hormones, supported the AC system might play a central the stimulus for decidualization.
activated after a decidual et al., 1983; Bekairi et was observed in rats
by
Day 4, induction,
that activity of AC decidual induction
1983).
suggested that decidualization
and mice. in our laboratory (AC), the enzyme
College
the the
protein.
(Km)
only on decidual
(cAMP).
1979;Kennedy,
of cAMP, was applied (Sanders This activation
Present Address: Riyadh, Saudi Arabia. Address: Riyadh, Saudi Arabia.
after after
optimum for the enzymatic reaction (30about 7 to about 16 pmoles/minmg proafter trauma. Calculations of the apparent
data suggested
in the
5 min and maximal of a decidual-inducing
1977,
workers have in initiating
in rats work cyclase
the formation stimulus was al., 1984a,b).
3’,5’-monophosphate
et al.,
60s
influences.
is evident within the application
(Rankin
studied
constant
calcium
The
alteration
Among the earliest biochemical events associated with the decidual cell reaction (DCR) in the uterus the mouse and rat is an increase in the concentration This response 15 mm after
were Within
Ea (20-30#{176}c) from 4.1 ± 0.8 to 13.0 ± 1.8 kcal/moldeg in the Qio (20-30#{176} C) was measured. After trauma, AC
INTRODUCTION
of
rat
estrus).
or in the temperature was increased from 7.5-8.0 to 6.5-9.5
inhibition an
=
6 to 16 pmoles/minmg
after trauma.
followed
in the 0
of activation (Ea) revealed an increase in trauma. A similar but less dramatic increase
creased in
(Ac) (Day
activity of AC increased from were measured in the apparent
0.3 mM), in the Hill coefficient 40#{176}C). However, the maximum tein and the pH optimum was energy after
cyclase
progesterone
tion Hill
5’-triphosphate pH optimum,
energy, coefficient.
established mild 100
trauma
Q10,
for
the
to the
(ATP), temperature
inhibition by In addition, activation uterus,
of
maximum optimum,
velocity activa-
calcium a time
ion, and course
cyclase
following
to determine
how
the was
rapidly
a
ADENYLATE
the system active state.
shifted
from
the
less
CYCLASE
active
to
the
AND
more
DECIDUAL
serum albumin, and 0.05-2.0 mM Reaction
MATERIALS
AND
METHODS
Animals Adult Co.,
female
Holtzman
Madison,
mentally
WI)
rats
were
controlled
(200-300
caged
quarters
g; Holtzman
individually (23
±
in environ-
2#{176}C) under
a light-
ing schedule of 14L: 1OD fluorescent illumination with the midpoint of the light phase set at 1200 h. Food and water were provided ad libitum, and reproductive recorded
cyclicity for at
was least
monitored 10 days
by before
vaginal animals
smears were
used in an experiment. Pseudopregnancy was induced in the rats by stimulation of the uterine cervix on the morning of proestrus and estrus; Day 1 of pseudopregnancy
was
filtration
defined in the
as the
vaginal
first
smear
day
of leukocytic
following
in-
tal
Uteri were group on
obtained selected
from days
The between
were
removed allowed
rats were weighed 0900 and 1200
quickly, to
and mesentery. were traumatized
3-6 rats per of the estrous
transferred
cool,
and
to
stripped
an
ice-cold
of adherent
In a second set of experiments, in situ on Day 4 of progestation
massage 1984b). posttrauma,
with a blunt instrument At selected intervals ranging the tissues were quickly
cooled,
as above.
All
tissue horns,
were was
1-cm izing
segments, and tube containing
7.4. The uterus/tube)
and killed h. The uteri
subsequent
fat uteri by
(Bekairi et al., from 5-120 s removed and
manipulations
of the
done at 4#{176}C. Each uterus, including both weighed on a torsion balance, cut into
tissue for
placed in a Kontes #22 homogen5 vol of 50 mM Tris-HC1, pH
was 2
homogenized mm and
in Tris assayed as
buffer (1 described
below. Adenylate
Cyclase
Activity of AC analysis as described Bekairi et conversion contained,
was measured by previously (Sanders
radiochemical et al., 1977;
al., 1984b), using the rate of [a-32 P] ATP to cyclic [32 P] AMP. The AC assay mixture in a final volume of 0.2 ml, the following:
15 mM Tris theophylline, phosphokinase,
HC1 (pH 7.4), 5 mM MgC12, 7.4 mM 14 mM phosphocreatine, 30 ig creatine 1 mM cyclic AMP, 0.08% bovine
preincubated
cpm), for
30
s
and
reactions
of and
the
et al., 1984b). of Schacterle albumin pmoles
were
100 pl of 0.75 M HC1. measured as described
by
Labeled previously
cAMP was (Bekairi
as a standard. The results are cAMP formed/min#{149}mg protein,
presented
as means
Selected
comparisons
using
Student’s
Effect
±
standard
errors
between
means
the
expressed and data of
the
were
as are
means. analyzed
t-test.
of Temperature effect measured
of by
temperature the use
from
on of
metestrous,
1.0-mM single points
concentration
of
homogenate of a series.
crine
condition
ATP
and
replicated
for
each
transformed to Arrhenius vation energies and Q10
Day
1 and
were run for 10 mm at 4#{176}C and 55#{176}C using a
for at least The determination
was
homogenates
the activity of AC uterine homogenate
proestrous,
Day 4 rats. All incubations selected temperatures between
ent
terminated
Protein was determined by the method and Pollack (1973), using bovine serum
3-6
an five
of
a
temperature for each endo-
times
replication. plots. values
aliquot
with
The
From these (20-30#{176}C),
differ-
data data were
were actical-
culated. Effect
of pH
The effect of pH on the activity of AC was measured by use of uterine homogenate preparations from rats during Day 4. Uteri from selected rats were traumatized in situ before preparation of the homogenate. Controls were nontraumatized. All incubations were
for
10 mm
tration
of
ATP.
5-10
and
was
at 30#{176}Cand The
Effects
of Calcium
pH
of
maintained
cine-piperazine-HC1
Assay
10
addition isolated
The
experimencycle and
were
X
tions),
preparations
pseudopregnancy. by decapitation plate,
mixtures
(1.2
of the reaction by addition of 50 .il (“.,l.l mg protein). Incubations for 10 min at 30#{176}C (routine condi-
estrus.
Preparation
[a-32 P] ATP ATP.
before initiation of the homogenate were performed
was Tissue
101
STIMULI
buffer
used the with
instead
a 1.0-mM media 50-mM of Tris-HC1.
activity uteri
of AC were from rats on
progestation. The homogenates were media containing concentrations of between 0 and 10 mM. All incubations at 30#{176}C and
from
glycyigly-
Chloride
Measurements of control and traumatized
min
concen-
ranged
pH 7.4.
made in Day 4 of
incubated in CaC12 ranging were for 10
102
SANDERS
ET
AL.
Reagents [a-32 P] ATP was MA,
(specific
activity
obtained from New and [8-s H] adenosine
England 3’,5’-cyclic
(specific acitivity Schwartz-Mann,
23 Ci/mmole) Orangeburg,
was
ion-exchange
purified
reagents disodium
by
and
drugs used salt, creatine
phosphate,
cyclic
(Sigma alumina Cleveland,
and
(Mallinckrodt,
and
St.
NY.
Nuclear, Boston, monophosphate
a
purchased from Cyclic [3 H] AMP
E
their sources phosphokinase,
and
Company, I (ICN
OH);
was
bovine
+Trauma
.
The were ATP creatine
serum
St. Louis, Nutritional manganese
Louis,
Ci/mmole)
chromatography.
AMP,
Chemical activity
4.0-40.0
E 0
albumin
0
MO); neutral Biochemicals, dioxide
12
E
10 Control
0.
8
powder
MO).
4 C) I.,
RESU LTS Time
Course
for
Traumatic
Activation
of Uterine
The initial experiment was designated the rapidity of activation of AC after the of a mild traumatic stimulus to the uterus of progestation. a significant
The (p