Responses to a Decidual-Inducing Stimulus

BIOLOGY OF REPRODUCTION 35, 100-105 (1986) Adenylate Cyclase in the Rat: to a Decidual-Inducing Stimulus’ Uterine Responses R. B. SANDERS,2 F...
4 downloads 2 Views 986KB Size
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

Suggest Documents