Polyamine biosynthetic decarboxylase activities following estradiol-17β or estriol stimulation of the immature rat uterus

3168

609

POLYAMINE ESTRAOIOL-178

Lynn

A.

BIOSYNTHETIC OR ESTRIOL

Lavla’,

*Departments

Sidney

of Internal University

‘To

OECARBOXYLASE STIMULATION

J.

Stohs’,

and

Medlclne Nebraska Nebraska Omaha,

of

Kansas Wesley Wlch

Henry

Lemon’

M.

Chemistry

66105

should and

School Medlcal Ita,

ACTIVITIES FOLLOWING THE IMMATURE RAT UTERUS

and Blomedlcinal Medlcal Center

of

whom correspondence Department of Obstetrics

University

OF

of

be addressed: Gynecology

Medlclne Center

Kansas

-

Wichita

67214

ABSTRACT Followlng estradlol-178

a single (E2) Into

decarboxylase

(ODC) It

postlnjectlon. remalned

elevated

When either Increased’ 10 hours. doslng to

control

pg

E3.

to

After

an

hours hours).

and

a

hours

of which InJectIon

Decreases decrease .In

an hours

5.0

ug

E2

In ODC nuclear

for

actlvtty estrogen

0.5 lferatlng

ug

E2

of

ODC actlvlty 0.05

4 and ,levels. and

actlvlty after decreased ug

E2

10

Is slmllar may be due

or

0.5

had and levels. a 4 hour by 14 hours.

Increased the

by

actlvlty

.ug E2 control

had of

and

hours. levels

only levels

actlvlty remainder

16

actlvlty 0.5 to

E3 stimulated to control

following receptor E2 InJectIon systems

by

(SAMDC)

SAMDC

4 hours by 6 hours

was InJected, to control

InJectton of then decreased

the

0.5 ug ornlthlne

levels

either

decarboxylase

elevated

seen here followlng seen In other pro1 mechanism.

wlth

of

peak at levels

levels same time

ug E2 or 0.5 ug SAMDC decreased of

remalned

0.05 ug decreased

treatment

followlng until 16

0.05 tlme

or then

a

control

to lntermedlate occurred at the

methlonlne

by 4 elevated

to

fE3) peak

followlng

S-Adenosyl

An InJectIon peak after

returning

decrease ug E2

InJectIon rats uterine

female

increased to to lntermedlate

0.5 ug estrlol a 4 hour ODC

levels

Increased remained

actlvlty decreased until

The 0.5

wlth

lntraperltoneal Immature

by

experlment

4 (16

hours may reflect The ODC actlvlty In to

tlmlng to a common

that

INTRODUCTION The

polyam

lnes

Volume42,Number6

have

been

shown

S=mBaOmD

to

be

lnvo

lved

In

cellular

December 1983

S

610

growth

and

has

obtained

a

catlons

synthesis

study

to

of

been

precise

under

linked

nature

ascertain

systems

estrogen-stimulated

polyamlne

been

dlfflcult

number

have

the

a

to

protein

uterus

Is

of

as

evidence

variety

of

this has

been

condltlons.

synthesis

and/or

DNA

(l-4). The

well

blosynthetlc for

estrogen The

some

a

lnjectlon

uterus

1s

questlon

Ideal

up

number

of

diverse

system

In

activltles of

(e.g.,

made

of

an

decarboxylase

characterized

following

Is

been

from

These

but

dlfferentlatlon,

Involvement

WDEOXDI

which

to

because

parameters

of

It

has

growth

5-9).

several

cell

responses

populations.

to

E2

Wh I I e

stlmulatlon,

there

separate .

laboratorles dlvlde E2

have

provlded

relatively

pattern

at

function

least

objective of

the of

that

synchronously

stlmulatlon, The

evidence

of

over the

polyamlne

time

a

In

response

24

hour

studies

NE)

Three were

from

Sigma

week-old Injected

Chemical

with

fraction. uter l/group.

and .uM

The Snyder

contalned The

Each

AND

group

(St.

was

(PLP)

to

measure

the

actlvltles

as

pH

in

al

4.0

I

Inc.,

Omaha,

(containing (E2) or estrlol various times thereafter. Into a homogenlzatlon

the 10,000 utlllzed

placed

for

(Sasco,

saline

glycylglyclne,

ut I I lzed studies

and L-Swere purchased chemicals were MO).

Rats

ODC assay was performed according (15) with mlnor modlflcatlons.

pyrldoxal-S-phosphate

was

LOUIS,

ml normal either l7B-estradlol dlslocatlon at removed and placed mM

of

METHODS

0.10

study enzyme

InJectIon

(6.10).

here

Sprague-Dawley

100

Each uterine

cells

InJectIon.

Co.,

female I.P.

lumlnal

sl’ngle

tsp. act. 52 mCi/mmole) (sp. act. 54 inCl/mmole) (Boston, MA). All other

ethanol) with or wlthout and sacrlflced by cervical Whole uteri were quickly buffer which dlthlothreltol.

a

decarboxylase

MATERIALS

purchased

to

reported

estrogen

DL-Ornlthlne-l-14-C adenosyl methlonlne-l-14C from New England Nuclear

and

perlod

blosynthetlc

followlng

stromal

3

ml

of

to

the

The routlne

7.4 x

and

5.0

10% (E3)

mM

g supernatant groups with

2

buffer. method

assay assays,

of

Russell

contained and 3.8

190 nM

a

S 1-14 C-DL-orn the

Ith

method

of

assay contalned methlonlne. 0.40 0.40

ml ml

of of

inc. Russell

The SAMDC and Taylor nM putresclne volumes for

organ

supernatant organ supernatant

inactivated SAMDC assays

at

concentration SAMDC actlvlty parallel to

and lncubatlon was hlgher, the saturated

InJectIon

of All

70’ were

0.5

Ng

by

utll izlng consldered

or

to The

nM lC- S-adenosy I were 0.52 ml, Including Controls contained which

had

and

ODC

and

buffer alone. to changes In enzyme Furthermore, both the

time. actlvlty In the

patterns 24 hour

up

been Both ODC

were period

found following

to

to

be

E2.

were corrected background Slgnlflcant IO-20%.

ly

3.8 assays

fraction. fraction

for 30 mlnutes, I lnear In response

and

.typlcal determlned

both

but assay

611

was performed according with mlno;4modlflcatlons.

and

data

efflclency

assay (13)

3.8 Total the either

waassoxxam

Duncan’s

for llquld sclntillatlon the coefflclents of differences between

and

Multlple

the StatIstIcal statlstlcally

Comparlson

Analysis slgnlflcant

Test

System when

counting varlatlon points

were were

followlng Differences

ANOVA were

(SAS). p < 0.05.

RESULTS A

sln,gle

immature

I.P.

rats

InJection

stimulated

decarboxylase

(ODC)

InJectIon.

Actlvlty

hours ODC

and had

of

then

to

In

(Table

elevated

returned

fig

Increases actlvlty

remaIned

0.50

uterine I)

decreased at

control

estradlol-17B

peaked

by

4

hours

after

levels

levels I8

hours

lntermedlate

Intermediate

levels

Into

ornlthlne

which to

(E2)

until after

by 16

6

hours.

E2

admlnlstratlon. S-Adenosy

I

meth

Ion

Ine hours

markedly

elevated

by

4

actlvlty

appeared

to

be

slgnlflcant

differences

hour

levels.

after

the

SAMDC admlnlstratlon

decarboxy

be

noted

decl

lned

-E2.

act

(Table

however,

could

of

(SAMDC)

postlnJectlon

blphaslc;

activity

lase

no between

to

control

Iv Ity

I).

was

The

SAMDC

statlstlcally the

4, levels

6,

8, by

and I8

IO hours

S

612

TIFDEOTDE

Table Uterine

Supernatant

ODC A

Time

After (Hours) 0

Actlvlty

Single

As

I.P.

A

ODC

InJection

Of

Time

Following

E2

Actlvlty

SAMOC

(dpm/uterus) 8610 + 1170

Actlvlty

(dpm/uterus) 2560 +

310

7370

5960

+

6 8 10

31430 27770 34130

+ + +

1700 4360 6620

5880 5610 6640

+ 3 +

150 490 430

14 16 18

26300 21910 11020

+ + +

3890 2560 1870

6570 5550 3720

+ + +

330 180 500

20

6240

2

760

3580

-,

330

24

5420

+

190

2140

+

720

are

the Three

Significance

was

mean to

+ S.E. of four assays

determlned

by

Test (p < .05). ODC Actlvlty: 0=18=20=24;

results were ANOVA

from performed

Uterine

After (Hours)

Single

by

not 4=10;

groups each

on

1190

of group.

Duncan’s

two

Multiple

slgnlflcantly 6=8=10=14=16.

dlfferent, SAMDC

4=6=8=10=14=16.

ODC

Supernatant A

three

followed

Time points 0=18=20=24;

Table Actlvlty

2 As

InJection

I.P.

of

A

Function

5.0,

Of

0.05, E2

InJectIon

Or

Time

0.005

Dosage

5 .o

Followlng ug

Actlvlty

E2

(ug)

0.05 ODC

0.005 (dpm/uterus)

0 4

650 10830

+ 2

60 1050

650 9450

+ +

60 340

650 2840

+ +

60 990

6 10

7180 7200

+ +

1670 710

7470 2550

+ +

360 220

3160 2180

+ +

750 180

14 16

6240 3120

+ +

760 720

2810 2060

+ +

110 220

1670 1200

+ 2

60 180

Values 1 each.

Signlflcance Comparlson are

-ug

+

Comparlson are I I sted. Actlvlty:

uter

Of

0.5

43 170

Values I each.

Time

Fun&Ion

InJectIon

4

uter

1

are Three was Test

I lsted.

5.0

the

mean

+

to four determlned (p < .05). Jig:

S.E. of results were perfor a ssays by ANOVA followed Time

6=10=

1 4.

polnts 0.05

from ed

groups of each group.

by Duncan’s s gnlflcantly

not Jig:

three on

4=16.

lO=

Multiple differen

0.005

Jig:

0=4=6=10=14=16. Many estrogen had

growth

parameter

receptor returned

I evels

occupancy. to

control

levels

change

as

Since

ODC

and

by

approximately

a

function

SAMDC

of actlvlty

16-24

nuclear patterns

hours

post-

two

t

S TmEQrDI InJect

Ion,

22-24

day

OOC old

Injection 2 not

peak

occurred

to

E2

and

the

Followlng

Increase

the

0.05

ug

0.005

ug

dose

to

0.05

an the

4

5.0

4

to

pg

E2

only

of

0.005

the

Time

Supernatant A Single After

I.P.

SAMDC Activity InJectIon

E2 ODC 1090 1960

L +

190 260

6 10 14

2280 1990 2620

+ + +

220 160 180

16

2760

2

320

Values uter I e&h. Slgnlflcance

are Three was

Comparlson are I isted.

Test

0.005

6=10;

Llg:

5.0

the

mean

to four determined (p c ug:

+

S.E.

hour

0=4=14=16;‘-4=6=10=14=16.

peak

ug

E2,

ug SAMDC

activity

activity

of

Act

Of 0.005

Dosage

was did

Time .ug

not

Followlng E2

(Ng)

0.05 (dpm/uterus)

lvlty

0.005

1090 2420 2220

+ + +

190 170 80

1090 1820 2010

+ + +

190 150 280

1630 1690 1590

+ + +

50 100 30

2060 1370 1280

+ + -+

50 160 200

results

from

assays were performed by ANOVA followed

.05). Time 4=6=10=14=16.

0.005

InJectIon.

SAMDC

As A Function 5.0, 0.05,

Of

5.0

0 4

5.0

or

3

InJectIon

(Hours)

0.05,

level. Table

Uterine

hour

appeared

followlng

E2

4

slgnlficant).

of

4

ug

the

actlvlty

5.0,

hours

In

blphaslc

Only

not

InJectIon I6

shown

appeared

SI lght was

the an

are

dose.

by

an

for

hour

results

ug

(but

in

following

and

doslng

injection

maxfmal

The

dose.

After

monltored

period

E2.

the

followlng

of

hour

prolonged

lowlng

elevated

Injection

fig

fol

different.,

remained an

0.005

were

16

was

patterns

slightly

the

activity

following

actlvlty

actlvlty

seen.

ODC

following

were

After

0.05,

3.

patterns

during

slgnlflcantly)

occur

SAMDC

actlvrty

uterus

5.0,

and

(but

SAMDC

rat

of

Tables

and

613

polnts

not 0.05

three by

groups

on each Duncan’s

signlflcantly Aig: 4=6;

of group. MultIpIe dlfferent

10=14=16.

two

S

614

WDEOXD=

Table Uterine

Supernatant

OOC A

Actlvlty As I.P. Injection

Single

Time

After (Hours)

4 A

Function of 0.5

Of ug

(dpm/uterus)

6 10 14

3980 20100 13280

+ + 2

320 2870 2600

7680 3430 3400

+ + +

800 1040 200

3080

-+

140

16 24

are

of results from three groups of S.E. assays were performed on each group. by ANOVA followed by Duncan’s Multlple Time polnts not slgnlflcantly dlfferent

are the mean + Three to four was determlned Test (p < .05).

I Isted:

Uterine

Supernatant

SAMDC A

Time

Activity

Single

After

I .P.

.

5 As

A

lnjectlon

Function of

0.5

of ug

8 10 14 16 18 20 24 are

An increases SAMDC

:

+

S.E.

elevation

of

of uterine (Table increases

results

from

6=8=10;

0.5

ug

wet

weight 1)

1010 1980

+ +

100 200

1590 1480

+ +

140 160

1420 1230

+ +

70 150

1100 1020

+ +

90 80

1040 1170

+ +

40 100

three

groups

of

two

assays were performed on each group. by ANOVA followd by Duncan’s Multlple Time points not slgnlflcant ly different

0=14=16=18=20=24;

InJectIon In

effects-maxlmal

mean

Three to four was determined Test (p c .05).

Signlflcance Comparlson I 1 sted

the

Following

SAMDC Actlvlty (dpm/uterus)

lnjectlon

4 6

Values each.

Tlme

E3

(Hours) 0

are

two

0=14=16=24. Table

uteri

Followlng

ODC Activity

InJectIon

0 4

Values uteri each. Slgnlflcance Comparlson

Time E3

E2

and In

10=14=16=18.

stimulates (5),

the

protein both

both first

synthesis 24

hour

DNA

tlearly” ODC (7) synthesls

effects-4 act1 and

vlty

hour peak

and

“late** (6)

and

mltotic

S lndlces

(6)

along

actlvitles

for

(estrlol) Increases

test

the

to

In

period

16

of

a

DDC

activity

actlvlty

was

results

patt

peak

at

resul

shown 4

hours,

injected

erns

were are

Table

an 5.

declined

then

on

22-24

monltored

for

In

a

Table

separate of

actlvlty

fig

Is,

E3

only

4

(5).

To

uterine

ODC

day-old

rats,

a 24

hour

4.

Only

exper

lment

0.5

SAMDC

0.5

that

(5)

InJectIon

SAMDC

of

and

synthesis

Into

In

ODC

InJectIon

protein

shown

noted.

both

effects,

and

was

followlng In

An

estrogen-E3

ts

was

measured

are

weight

E3

of

uterine

acting

Ng

peak

1).

“early”

short

The

elevation

(Table

wet

actlvlty

thereafter.

. hour

hours the

0.5

ODC

continued

uterine

patterns, uterine

the

only

effect

activity

a

up

produces

hour

and

with

15

WDEOTDm

ug

the SAMDC

E3.

Increased

4

The

slightly

to

thereafter. DISCUSSION

Whl rat or

le

enzyme

uteri at

for

the

or

entire

IO

polnts E2

ODC

patterns

first

time

castrated

reported for

the

single

adult

activity

time

hours

(13).

no

Jg

one

E2

to

our

Immature

InJectIon

(11)

(12,141 knowledge

followlng

actlvlty

for

InJectIons

patterns

enzyme

reported

0.5

various

actlvlty of

been

followlng

followlng

InJected

SAMDC

have

or has

estrogen

elevation

In

In

InJection

the

Immature

rat

uterus. The 0.5

fig

E2

hours were

SAMDC

actlvlty

appear

postInJectIon not

other

to

patterns be and

slgnlflcantly

times

peak

at

4

Thls

uterine

(Table hours

reported

blphaslc, a

second

different 1).

The

ODC

postInJectIon ODC

actlvlty

then pattern

here

with

a

peak

at

from

enzyme

act

Ivi

first 10

y

fel (Tab

after

hours,

but

peaks

these measured

had

followfng

of by

a

4

at

promlnant

Intermedlate 1)

InJectIon

occurrlng

actlvlty

pattern to

e

peak

an

levels. a

single

0.5

S

616 pg

InJectIon

of

regenerating

Therefore,

It

changes This

estradiol-178

rat

synchronously

nhlch

actlvlty

HTC

appears

that

an

Is

following

growing

In

that

and

(E2)

Ilver

occur

suggests

S-=EOXDI

partial

cell

followlng

the

other

the

E2 systems

In

to

that

(16)

stimulation

(17).

uterus

stimulated I ead

seen

hepatectomy

stimulated

mechanism

increase

slmllar

Ing

prollferatlon

the

and

In

undergoes to

to

In

both

proliferate. the

(6,18)

elevated

Is

ODC

slmllar

In

both

the

uterine

systems. Nuclear target

estrogen

cell

InJect

nucleus

Ion

of

levels

begln

nuclear

peaked

E2

rodent

to

decl

Ine

by

In

followlng

which

estrogen

suggests

that

nuclear

hours

followed

by

levels

(21,22).

receptor

after

In

uterine 5-8

estrogen seen

at

either 4

elevation

seen levels

of

doses

these

actlvlty

0.5

hours

receptor

nuclear

InJectIon

an

after

of

The lel.

ODC

I

0.05 to

InJectIon

to

control

estrogen

of

#g

after

an

receptor

a

0.5

the

on

second

gg

E2

actlvlty

amplitude increased

ODC of

Injected, ulthout

Ng by

(23).

of

species and

0.5

levels

here

rate

was levels

of

receptors. as

E2

control

estrogens

monitored

mul tlple

duratl

or

an

these

estrogen

of

E3 fel

fall

such

presence

,ug

then

patterns

Involved,

paral

ug

hour

dence

evl

elevated

smaller The

ODC

may

reflect

ODC

actlvlty

phenomena. When

of

0.5

patterns

Is

is

are

f irst

the

There

Increase

actlvlty th

wlthln

(19,201.

Injection

I evels

receptor

E2. 6-8

reflect

However,

other

estrogen

after

suggests

directly

prolonged

Nuclear hours

Thls

synthesis

the

InJectIon that

changes factors

and

degradation

SAMDC

actlvltles

ODC In

may

levels also or

be the

ODC. of

ODC

and

dramatlcally

and

contained

were a

not

S lcant

slgnlf 0.5

of

E2

JJg

These

of

while

E2

NOTE: reported 10 ug

In

In Is

E2

be

at

but

16

of of

they

nearly

as

dramat

be

a

also The

an

report,

Ic.

signal

contlnulng InJection

another

of ODC activity uterus (24).

of

of

5.0

divergence.

metabolism

this

I on

may

followlng

pathway

submIssIon

similar patterns In Immature rat

not

metabolism. hours

Inject

an

were

pathway

evidence the

following

colncldental,

activity

further

hours Increases

polyamlne

SAMDC

Since

4

activity

may

divergence

Increase

activity

SAMDC

differences a

ug

peak

617

TDEOXDI

group

followlng

has

the

InJection

of

. ACKNOWLEDGEMENTS Thls work was Meeting, 1961. under Natlonal

presented LAL Cancer

Unlverslty

Nebraska

of

Thanks critical Mrs.

to Char

Or.

McCann of

Y.

the 63rd Annual Endocr supported as a teaching education grant #CA 19623

Ine

Society

fel -05

low at the

at

Medlcal

Peter

readlng I otte

In part partlally Institute

was

Center. of

Merrell

the manuscript. Hersom-Col I Ins

Dow The grateful

Is

Research

Center

for

secretarial assistance ly appreciated.

of

REFERENCES I.

2.

Loftfleld, Polyamlnes

R.B., Elgner, In Biology Dekker, Inc.

M. eds.), WI I I lams-Ashman, 8101.

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