Progesterone in the uterus

731

PROGESTERONE V.

CORRELATION WITH

OF

THE

THE

IN

IN VITRO

PROGESTERONE Dieter

Department

UTERUS.

PROGESTERONE

BINDING

Egert of

THE

and

of

RAT

Heinrich

Obstetrics

University

IN

and

METABOLISM UTERUS.

Maass

Gynaecology

Hamburg

Hamburg-Eppendorf,

Germany

(FRG)

5/19/75

Received:

ABSTRACT

3 H-proIncubations of rat uterine segments with varying gesterone concentrations were performed to study the The radioactivity of the hormone uptake by the tissue. uterus and the nutrient medium were plotted in form of a the binding capacity of the SCATCHARD plot. Additionally, uterine cytosol was measured. In both systems, the hormone was found to be associated with two components which differ from each other in their association constants. The progesterone metabolism occuring at a hormone concentration of 10-6 M and more in the incubation medium is discussed with respect to the affinity and the capacity of the hormone binding components. INTRODUCTION The

occurance

ically

and

demonstrated

paper

hormone observed

we

(51,

gesterone nutrient

proteins

binding

unspecifically,

thoroughly

ing

of

found

metabolism medium. of in

10

-6

on

Only M

uterine

in

the

(l-4).

As

a dependence the

hormone

progesterone, rat

uterus

specifhas

been

described

in

the

of

in

vitro

the

concentration

with

concentrations

more,

progesterone

metabolites

tissue.

Furthermore,

no

and

of

preced-

in

prothe

labelled

significant

were

S

732

conversion

of

detected latter

progesterone

under

in

results,

metabolism

in

the

experiments gesterone one

to

vivo

it

as

was hormone. were

by

cytosol

in

rat

well

as

limited In

by

order

that in

vitro

the to

and

Considering

the

capacity

of

the

this the

the

progesterone low

test

be

hormone the

proteins

hypothesis, uptake

binding

of

of

pro-

progester-

proteins. MATERIAL

AND

METHODS

steroid. 1. Radioactive 1,2-3H-progesterone obtained from Radiochemical Centre Amersham for all experiments and purified before use

(5)*

could

(at

to measure

segments

uterus

(6,7).

supposed

performed

uterine

the

conditions

was

vivo

concentrations) binding

WDEOTDI

(S.A.= 1 Ci/mM) was applied as described

-

2. Buffer. Tris-HCl-buffer containing 0.01 M Tris, 0.001 M EDTA and 0.25 M sucrose (pH = 8.4). This pH-value was chosen because McGUIRE and BARISO (2) found the pH-optimum of progesterone binding at 8.4. 3. Chemicals. Norit A (charcoal) was obtained from Serva, and dextran T 70 from Pharmacia, Heidelberg, Germany, Uppsala, Sweden. Selecta filter paper No. 595 was obtained from 4. Filter. Schleicher & Schiill, Germany. $. Scintillator. Insta-gel from Packard Instrument, Frankfurt, Germany. 6. Centrifuge. Spinco L and Spinco L2 65B, Beckman InstruMiinchen, Germany. ment, TRI-CARB 3380 with an 7. Liquid scintillation counter. absolute activity analyser from Packard Instrument, FrankGermany. furt, 8. Protein measurement. The protein amounts of homogenates after preparing a cytosol) and cytosols were determined by the method of LOWRY et al.(8). 9. Animals. Female Wistar rats were used in all experiments and pretreated with estradiol-benzoate in the same manner as mentioned in the preceding paper (5). lO.Incubation of uterine segments. Uterine segments were incubated with labelled progesterone and the subsequent homogenization was performed in the same manner as described previously (5)._;Sh e progesterone concentration applied ranged from 5 x 10 to 5 x 10-6 M. The tissue powder obtained from uterine segments was suspended in Tris-HCl-

S

S--EOID-

733

The absolute radioactivity in the homobuffer (pH = 8.4). genate and in the nutrient medium were directly measured by a liquid scintillation counter with an absolute activity anaiyser. adsorption method (9) Uterine cytosols were 11. Charcoal prepared with Tris-HCl-buffer (pH I 8.4) from uterine segments as above mentioged and then centrifuged at each containing various 105 000 x g for 1 h at 0 C. Vials amounts of 3H-progesterone, dissolved in 0.4 ml chlorowere heated to 40°C to evaporate the organic solform, 0.5 ml aliquots of cytosol with a After cooling, vent. protein concentration of 1 mg/ml were filled into the The incubation mixtures were allowed to stand at vials. Thereafter the charcoal suspension was ad4'C over night. ded to the cytosol incubates. The mixtures were stirred then centrifuged at 1800 x g and filtered to for 45 min., remove small charcoal particles. The filtrates were measured to determine the radioactivity of protein bound proThe charcoal sediment was treated by agitation gesterone. with scintillator at room temperature over night, centrifuged and the extracted radioactivity was measured to determine the amount of progesterone not bound to protein. Two charcoal suspensions were used in different sets of experOne milliliter of suspension A (containing 250 mg iments. charcoal and 25 mg dextran T 70 in 100 ml Tris-HCl-buffer, pH = 8.4) was used in the experiments studying progesterone binding to cytosol proteins. Three milliliter of suspension B ( containing 500 mg charcoal and 50 mg dextran T 70 in 100 ml Tris-HCl-buffer, pH = 8.4 ) were applied in the experiments to determine the saturation of proteins binding progesterone. RESULTS of

3H-progesterone

after

incubation

1. Uptake One

hour

estrogen-primed tissue

and

rats,

in

the

concentration

5 x

to

1O-1o

gesterone culated The

5 x

(in: to

labelled

of

the

nutrient

gesterone

lO-8

moles)

a protein hormone

uterine

uterine

the

. The

found

segments in

were

of

the

uterine

moles)

of

the

medium

amount

in

taken

The

determined

pro-

ranged

radioactive

per in

from pro-

tissue

1 gram

from

uterine

measured.

nutrient

concentration (in:

segments.

radioactivity medium

in M

by

was

cal-

1 liter.

the

nutri-

S

734 medium

ent

were

plotted

bound in

were

calculated

in

form

progesterone

the

amount

was

uterus of

used

(UT)

labelled in

TBEOXDI

the

‘1,O

to

of

1 liter.

a SCATCHARD

the

amount

and

instead

hormone SCATCHARD

in

of of the

The plot

values (10).

radioactive unbound

The

Instead

of

progesterone progesterone

nutrient

equation.

obtained

medium plot

in

the

(NM) Fig.

1

#x10’

0,:

I

1

1 2

UT I

H

3x10-9[M]

by rat uterine segments: Fig. 7 Uptake of 3H-progesterone of progesterone in uterine tisabscissa = UT, the amount sue (moles progesterone calculated to a protein concentration of 1 g/l). Ordinate = UT/NM. NM = progesterone concentration in the nutrient medium after incubation (M). the progesterone concentration ranged Before incubation, from 5 x lO-Jo to 5 x 10s8 M in the nutrient medium. Each point represents an average of 6 samples. a = First association component, b = second association component. The association constants and the interacting sites are given in Tab.1.

representing tissue

follows

UT NM

The

the uptake

k(n-

=

graphic

curved

the

of %-progesterone

the uterine

equation: k= association constant n = maximum number of interacting groups

UT)

presentation

line,

by

which

given

in Fig.1

demonstates

resulted

the association

in a of pro-

gesterone

to two components

in the uterine

segments.

This

curve

resolved

straight

according

to

was

the method

into

reported

two

by WEDER

lines

et al.(ll).

Line

the association

of progesterone

to the first

uterine

The

constant

this

tissue.

line

was

calculated

of interacting progesterone

sites

and

a higher

the

second

value

1.1

found

to be

to a second

capacity.

component

The was

the intercept

determined

gesterone

was

to be

was

found

M

The

of %-progesterone

A. Uterine

cytosol

-1

.

The

4.4 x 10 -13

in

from number

moles

the association

with

a lower

of interacting

to be

2. Binding

from

the with

1.8 x 10

association

-11

affinity

sites

slope

of

of

line b. The moles

constant

prowas

2.0 x IO6 M-l.

with

separated

component

calculated

to be

hormone

8

of the ordinate

per mg protein.

incubated

10

component

determined

p gives

number

calculated

was

x

Line

per mg protein.

of progesterone

line 2 and

association

5 represents

by

uterine

obtained

from

labelled

progesterone

from

the unbound

cytosol.

estrogen-pretreated

hormone

and by

rats

the bound the dextran-

charcoal

adsorption

was

in

used

progesterone

this

technique. assay

concentration

to 5 x 10 -7 M. The progesterone plot

(70).

(see

were The

The

materials applied

measured

values

graphically

amount

of

charcoal

bound

and

varied of

presented

suspension methods). from

bound by

progesterone

4 x

and

A

The 10 -9

unbound

a SCATCHABD (in:

moles)

Z,O-

45.

l,O-

0,s

SCATCHARD plot of components in rat uterine cytosol Fig. 2 binding progesterone: abscissa = B, amount of bound progesterone (moles progesterone calculated to a protein concentration of 1 g/l). Ordinate = B/U. U = concentration of unbound progesterone (M). Before incubation, the rogesterone concentration ranged from 4 x 10-9 to 5 x IO- s M in the incubation medium. The separation of bound from unbound progesterone was performed by charcoal suspension A. Each point represents an average of 8 samples. a = First binding component, b = second binding component. The association constants and the binding sites are given in Tab. 1.

s was

calculated

1 liter

and

in moles

per

amount

binding

resolved

into

binding

in uterine two

straight

the association

component

and

Association

determined

as mentioned constant

calculated sites

to be

was

was

1 gram

per

determined

found

of progesterone

lines manner

The by

;5rthat

constants

the method

as in Fig.

to the

the first 10 7 M-1

2.9 x

3.6

to be

x

to the

and binding

binding

and

10 -13

of a

first

binding

sites

were

paragraph.

The

component

was

the number moles

curve

I. Line

second

in the preceding

of

to the

resulting

of progesterone

line

component,

association

of

hormone

cytosol.

in a similar

et al.(lf)

represents

of unbound

the association

components

WEDER

concentration

1 liter.

2 illustrates

Fig.

was

to a protein

the

737

fIF1BBlOSDI

of binding

progesterone

per

mg protein. The

association

ponent

in uterine

and

a higher

The

association

number moles

cytosol

capacity, constant sites

progesterone

per

saturation

experiment, gesterone tration 3.0

of

studied

IO-%

mg/ml

found was

and

shows

was

second a lower by

5.0

com-

affinity

line 2 in Fig.

x

determined

binding

105 M-'

to be

1.5

and x

2.

the

10 -17

mg protein.

of binding

in which was

which

to the

is illustrated

of binding

B. The

from

of progesterone

proteins

the ratio

of bound

at the constant the protein

to 0.725

mg/

ml

was

measured

to unbound

progesterone

concentration (Fig.

3).

The

by an proconcen-

ranging separation

S

738 of

bound

the

from

unbound

B,

Fig.

a constant

3,

protein ml was

see

was

found

second

material

and

amount

be

1.9

The x

10

corresponds component

I

-11

I

both

1

also

technique

bound 1.5

As

moles the

performed

and

0.125 of

sus-

illustrated

in

mg

per

of

mg

protein

binding

per

proteins

progesterone

number

by

(charcoal

progesterone

saturation

to in

was

methods).

of

between

medium.

to and

adsorption

observed

incubation

protein the

progesterone

dextran-charcoal

pension

WXIEOXDI

per

binding

mg

sites

of

assays.

1

I

I

3,5 3,O 2,5 2,O 1,5 1,O 0,s mg protein /ml

J

Fig. 2 Saturation of progesterone binding components in uterine cytosol. At a constant progesterone concentration (10'6M) the amount of cytosol protein varied in the incubation medium. The separation of bound from unbound progesterone was performed utilizing the charcoal suspension B. Each point represents an average of 7 samples.

x

3H-progesterone

Saturation

of

x

1.8

4.4

by

x

x

8

10 6

10

Binding

1.9

x

10

5.0

2.9

rat

x

x

105

10-l'

IO -13

protein

x

1.5

/ mg

x

3.6

/

M-’

-1 10 7 El

uterine

progesterone

Ka=

moles

II:

:

(moles I

to

: Ka=

II:

I

constants:

binding

-11

component

component

sites :

component

component

Binding protein)

Binding

2. mg

Binding

Binding

progesterone:

lo-"

/

M-'

M-’

10 -13

progesterone

2.0

1.1

proteins

:

II:

I

(moles

Ka=

: Ka=

II:

I

cytosol

component

Association

3.

component

sites

Association

Interacting protein) :

component

Association

2. mg

component

Association

constants: 1. Association

rat

1. Association

by cytosol

3 H-progesterone

1

In vitro uptake uterine segments

of

TABLE

S

740 3 . Progesterone Festerone

the

similar

uptake

to

and

ond

of

the the

results

only

to

With

regard

progesterone lism

are

as

moles

tissue

as

1).

by

the

in

led the

progesterone the

found initial

to

uterine

under

the

in

paper

the

Tab.

from

the

in

was Fig.

somewhat

lower

or

of

than

total

binding

of

the and

was

conditions

by

of that

secshow-

binding

comparison

segments

by

experiments

sites

(5),

the

uptake

conclusion

vitro

2. At

the the

related

applied.

the

results

of

progesterone

metaboof

5 x

1O-'1

an

amount

of

2.5

x

observed

in

100

uterine

4.

a concentration

the

medium

nutrient

the

the

cytosol

experiments of

Therefore,

and

the

of

the

of

binding

sets

were

constants

types

saturation

progesterone derived

the

both

to

interacting

both

prothat

which

progesterone

The in

in

binding

were

the

preceding

in

the

cytosol

Also,

binding

compared

lo-'O

and

in

components

experiments,

which

the

binding

progesterone

against

pro-

demonstrated

protein

association

the

systems

Protein

the

the

The

in

uptake

to

of

measured.

agreed

both

progesterone

be

size.

(Tab.

of

can

component

value

progesterone

It

in

was

same

same

binding.

in

first

component the

vitro

experiments

in

segments of

were

in

the

studied.

experiments,

uterine sites

the

of

segments

components

the

to

results

other

uterine

were

binding in

relation

constants

each

into

proteins

M

the

association

uptake

in

metabolism.

gesterone

ed

binding

1 summarizes

Tab.

TDDOXDI

In

uterine

progesterone

Fig.

4 the

segments

mg

amounts

are

concentration

plotted in

the

of

s nutrient taken

from

Together one

Part

medium. the

with

of the data,

set of experiments additional

concentration

to 5 x 10 -' M .

I.

,

741

!EEIcOXDI

experimental

amount

, . , . *, ,

data,

medium

I”“,

concentration

7

’

lO-7 in nutrient

1

.

10-6

1.

the progester-

*

9

10 -9

from

moles

,‘“‘I

was

in Fig.

ranged

of 2.5 x lOBJo

10+ progesterone

in Fig.&,

represented

in the nutrient The

shown

per

,(V’.(

100 mg

@

WI

medium

Fig. 4 Uptake of 3H-progesterone by uterine segments. The progesterone concent ation in the nutrient medium ranged from 10-9 to 5 x 10' ii M,

uterine

segments,

amount found

of at

2.0 the

the

nutrient

The

maximum

in

x

10

Fig.

gesterone

to

corresponds

progesterone

2).

(Tab. of

mg

(SCATCHARD

1.5

(SCATCHARD

analysis of

to

The

10 of

-11

total

the

binding

progesterone

x

the

10

-11 of

mg

M

in

components

progesterone moles

pro-

bound

uterine

progesterone

per

to

progesterone

proteins

10"

the

amount

in

moles

of

an

protein,

5 x

of

1.8

to

mg

associated

be

components

x

per

analysis

observed

protein

saturation

progesterone

protein.

as

moles

moles

4,

medium

determined

IO"'

Fig.

concentration

1) was

per

from

progesterone

segments

gesterone

The

-11

amount

uterine

uptake,

taken

pro-

cytosol per

mg

binding,

was

protein

Fig.

2). 1.9

x

was

found

to be

protein

(Fig.

3

and

Tab.

in

the

nutri-

2). Up

to

the

ent

medium,

one

per

mg

which

described

uterine

In

present

in

labelled

ed

in

in

the

than

the

a recovery

10 -7

tissue Injection

of

endogenous of

1.75

10

in

M

the

in

in

moles

progester-

level moles

rat

of

uterus

Metabolites

in

a progesterone

amount vivo

the

nutrient

of

of

the con-

medium.

progesterone

application

a progesterone

-11

M

radiometabolites

at

hormone x

-11

only

the

after

10 -7

(5).

paper

found

5 x

10

detected

2 represents

uterine

hormone.

be

be

x

tissue,

preceding

than

Tab.

2.0

uterine not

could

higher

addition,

higher

could

segments

centration

in

5 x

of

corresponds

protein

3H-progesterone as

concentration

hormone

dose the

of 50

the times

rat,reeult-

progesterone

per

S

743

TI1EOXDI

I

I

J

+

+

744 100

mg

tissue

much

the

per

lower

binding

10"'

which

(6),

progesterone is

WEEOXDI

s

mg

protein.

than

that

components

moles

per

mg

corresponds This

necessary in

the

to

1.4

amount

of

for

the

uterine

x

lo-l2

moles

progesterone

saturation

of

(1.5

cytosol

-

1.9

x

protein). DISCUSSION

Two

binding

differ

from

components

are

each

in

progesterone. MILGROM et

This

and

al.(s)

other

who

their

result

BAULIEU

McGUIRE

two

in

the

affinity

agrees

(l),

detected

occuring

and

with

the

and

BARISO

binding

rat

uterus.

They

capacity

to

observations (2),

proteins

in

and

rat

by FEIL

uterine

tissue. The

association

presented

in Tab.

and

BAULIEU

who

reported

1.5

x

rat

uterus.

may

be

lo8

to

In

addition,

also pared

et

the

the (3),

al.

specific

binding

findings and

constants

differences

different

first

of

DAVIES

5.5

between

the

methods

for

x

RYAN 10

of the

this

(4)

7 and

receptor

values

used

MILGROM

and

progesterone

to

component

in

study

determination

constants.

and

data

a

second

a higher

gesterone The

for

The

the

FEIL

the

with

association

M-l

due

of

1 agree

(I),

of

ity

constants

capacity,

receptor on

the

revealed with

the

binding

ItI was

second

a lower first

component

the

so-called

reported

component affinity binding

with

by

I' unspecific

these

observed and

affinpro-

authors(1,3,4). in

a higher

component,

a lower

and

this

study

capacity, seems

to

comcor-

S

?FD81OXD-

respond

to the properties

scribed

by

the above

Additionally,

Such

posed

a correlation

vccur

only

paper

total

conclusion

that

protein

complexes

and

complexes

20u-hydroxysteroid between

dissociation

presented

(15)

aup-

content

and

in the uterus

of the enzyme of the

those

of

in Tab.

metabolism

concentrations

further

constants

higher

2,

does

than

by a correlation of the progesterone

The

constants

of pro-

MI~HAELIS

constants

of 'ju-hydrvxysteroid

1.0 x 10 -5

and

and

that

complexes.

lower Vice

component

10 -4 M

from

were

(12-I&),

binding

than

(5.0

cvn-

compvnent

the M~CHA~LIS

versa,

The

the association

the dissociation

progesterone

is even

dehydrogenase,

5D-reductase

1.0 x

calculated

illustrate

second

with

illustrated

a progesterone

dehydrogenase

of the second

here

are

enzymes.

constants

(2.0 x 70 -6 M)

stant

by protein

(4), who

proteins

the MICHAELIS

metabolizing

of steroid

stant

binding

is supported

the dissociation

constants

uptake

binding.

between

reported

binding.

the progesterone

at progesterone for

gesterone

of progester-

to protein

end RYAN

(51, which

to conclude

necessary This

by DAVIES

of the data

the preceding us

de-

rats.

comparison

leads

the uptake

is related

of progesterone

of pregnant

that

between

binder,

authors.

of the progesterone

was discussed

the amount

The

mentioned

segments

a modulation

binding

of the unspecific

we demonstrate

one by uterine

745

constants

the association

x lo5 M")

con-

is higher

S than

those

calculated

metabolizing

enzymes the

Therefore, gesterone ity

of

is the

enzymes

authors

previously

also

The

in

showed after

SAFFRAN

that

the all

the

This

is

true

previous

progesterone

the

binding

of

affin-

reaction. and

A

cor-

progesterone

(16)

for

of the

the

total With

and

by

these

rat

hormone

should

like

illustrating metabolism

(18),

in of

to that

uterus

of

should

were

per-

this

an

earlier

study

was

data

10

in

times possibly

presented

progesterone vivo

(6)

found

progesterone

after

same

study

progesterone

the

the

(5,6,17).

hormone of

it

under

experiments

amount

regard

progesterone

estradiol

labelled

evident

the

of

publications

maximum

becomes

progress

unbound

compared

with

amount

we

only

tissue

the

labelled

Thus,

enzyme

experiments

pretreated

in

the

al.

the

pro-

than

amount

that

it

to

higher

et

uterine

rats

2).

of

10 5 M-l).

component

metabolism

reported

metabolized

Finally,

the

experiments

(Tab.

the

by

x

level

for

vivo

than

paper,

in

in

the

1.0

second

saturation

influences

injection

smaller bound

of

and

constants

(17).

proteins

and

of

suggested

estradiol

4

the

progesterone

was

emphasized

10

progesterone.

available

binding

conditions.

to

be

between

with

x

MICHAELIS

5 times

the

relation

formed

the

of

above

will

be

(1.0

affinity

proteins

binding

from

approximately

progesterone

Since

TDEOIDI

in

cannot

this

be

application

(6). to mention the on

that

dependence

the

experiments of

progesterone

the

in

are vitro

binding.

S

747

w?DEOXDI ACKNOWLEDGMENT

Sonderforschungsbereich This work was supported by DFG, The authors would like to thank "Endokrinologie". Dr. F. Hoelzel for his help in preparing this manuscript in English.

34

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