Cortisol receptors in rabbit fetal lung

Vol. 47, No. 2,1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CORTISOL RECEPTORS IN RABBIT FETAL LUNG* George

Giannopoulos,**

Departments of Experimental the University Clinic,

Received

March

Shree

Mulay

and Samuel

Medicine and Biochemistry, Royal Victoria Hospital,

Solomon University Canada.

McGill

Montreal,

and

22, 1972

SUMMARY. Rabbit fetal lung nuclei contain macromolecules which have the properties of physiological receptors for cortisol by the criteria of specificity of binding and saturation of binding sites at low concentrations of the hormone The number of nuclear receptor sites is relatively low at 20 days of gestation, reaches a maximum at about 28-30 days of gestation and drops slightly after These results correlate well with previously reported changes in pulbirth. monary epithelial cell maturation and surfactant concentrations in rabbit fetal Preliminary evidence for the presence of cytoplasmic cortisollung extracts. binding components in fetal lung cells is also presented. INTRODUCTION. that

Numerous

the alveoli

substance,

forces

collapse

(l-3).

with

a high

its

involves

Present

lung

function both

appearance

pulmonary and supported

dence

that

surfactant

indicates

that

in

towards

is

Maturation

maturation

a lipoprotein

earlier

than

by Kotas

*

that

was suggested

expected (9).

These

and Avery

with

fetal

the

steroids

(8).

Preliminary

after

cortisol

eviinfusion

observations (lo),

lung

by Buckingham

were

who found

Supported by grants from the Medical Research Council of Canada and MT-1658) and the U.S. Public Health Service (No. HDO-4365) ** Scholar of the Medical Research Council of Canada. 411

to

of the fetal associated

of Liggins

by de Lemos et al

Copyright@ 1972,by Academic Press,Inc.

alveolar

the end of gestation

The possibility

by the observations

rabbits

alveolar

preventing

material

characteristics

cell

fetal

thereby

this

of gas exchange.

epithelial

reduces

(4,5).

changes

(5,6).

surface-active

presumably

respiration,

surfactant

have established

a highly

which

and biochemical

lambs was reported confirmed

with

lecithin

was present

studies

during

marked

as an organ

(7),

sequently

volumes evidence

anatomical

lined

surfactant,

undergoes

et al

of fetal

are

of dipalmitoyl

of pulmonary

may influence

lung

and experimental

lung

pulmonary

at low

content

The fetal permit

of mammalian

designated

surface

theoretical

(Nos.

that

sublung

MA-4744

Vol. 47, No. 2, 1972

maturity,

BIOCHEMICAL

as defined

monary

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

by accelerated

surfactant,

morphologic

was increased

The mechanism

by which

development

by glucocorticoid

cortisol

the interaction

of steroid

hormones

their

target

tissues

seems to be an early

action

(ll),

we have

examined

fetal

lung

with

maturation

specific

requisite

rabbit

pul-

treatment.

accelerates

Since

and increased

lung

receptor

step for

is not

in

known.

molecules

their

in

mechanism

the presence

of

of cortisol

receptors. METHODS.

Pregnant

gestation

under

fetuses

were

placed

white pentobarbitol

removed

in ice-cold

were

blotted

Hela

t9ssue

labeled

and immediately Eagle's

culture

The gravid

medium,

tissue

tissue

uterus

with

were

lungs

scissors.

CO2:

3H-cortisol

and the

were

excised,

The lung

minces

in 10 ml Eagle's

concentration

of 95% 02-5%

days of

was exposed

incubated

an appropriate

with

on at 20-30

The fetal

and minced

of the

containing

the

operated

of 3H-cortisol

Competition

studies

and a ten-fold

excess

were of non-

steroid. incubation,

0.01

M Tris,

was prepared

in

The homogenate of sucrose

pH 7.4,

three

Tris,

0.001

of

MgC12,

The purified

1 hour.

ml aliquots

phenylamine nuclear

method pellet

through

a glass four

with

Further

the crude

pH 7.4,

buffer,

nuclear

pellet

of the homogenate (12).

Separate

homogenate

homogenizer

were

M sucrose,

412

concentration was layered

The pellet

10 mins. M Tris,

0.001

for

with

at 100,000

in 3 ml of Buffer DNA determinations (in

triplicate)

3 ml ethanol

was

M MgC12,

was accomplished

by centrifugation

taken

0.32M

pestle.

in 10 ml of 2.4 M sucrose,

0.2 ml aliquots mixed

the

homogenate

0.01

was homogenized were

a teflon

of the nuclei

pellet

with

A) and a 5% homogenate with

diluted

at 1100 x g for

followed

times

of cheesecloth,

purification

nuclear

several

(Buffer

M and 15 ml of the

20 ml of 0.25

B).

washed

layers

A and centrifuged

times

were

p H 7.4,.buffer

using

to 0.25

(Buffer

by homogenization

minces

M MgC12,

was filtered

buffer

lung

the same buffer

was adjusted

washed

the

0.003

on top of 5 ml Buffer

fied

were

decapitated.

an atmosphere

by incubating

sucrose,

0.1-0.2

Hela

medium

at 3J” in

Following

then

rabbits

anesthesia.

and 1 gram aliquots

(45 Ci/mMole) performed

New Zealand

and,

0.01

x g for B and two by the

of the purifollowing

M

di-

Vol. 47, No. 2, 1972

BIOCHEMICAL

centrifugation,

the

and counted.

In other

in 3 ml of 0.01

supernatant

0.0015

the homogenate

was allowed

70,000

15 mins.

tered free

through

small

was also pH 7.4,

been

nuclear

M EDTA, 0.6 M KCl,

Aliquots

at 4 O for

pellet

pH 8.5,

30 mins

of the supernatant

G-50 columns

of cortisol

studied. buffer

buffer

and then (nuclear

fluid was homogenized (Buffer

C),

centrifuged extract)

at 4 o to separate

for

filtration

2 hours

on Sephadex

previously

described

=

40000

r

20000

-

were

the bound

components

of fetal

lung

homogenized

in 0.01

M Tris,

0.0015

fractions

(cytosol)

were

prepared

D) and soluble

the homogenates

incubated

to cytoplasmic

The tissues (Buffer

centrifuging

gel

10 ml of scintillation

the purified

to stand

Sephadex

with

at were

from

fil the

steroid. The binding

were

was mixed

experiments,

M Tris,

x g for

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

at 224,000 at 4O with

x g for

1 hour.

10m8M of 3H-cortisol

G-ZOO columns.

The methods

The cytosol

and liver M EDTA, by fractions

and fractionated of gel

filtration

by have

(13). Void

Volume

E c c 0 z

12000-= 10000

-

6000 8000

-

4000

-

2000

-

e L 28

-

125

100

Fraction

150

Number

Fig. 1. Gel filtration on Sephadex G-200 of rabbit fetal lung and liver cytosols and of serum pre-incubated with 1 x 10m8M 3H-cortisol for 2 hours at 4O. One-gram aliquots of tissues from rabbit fetuses at day 28 of gestation were homogenized with 3 ml of Buffer D. Following centrifugation of the homogenate at 224,000 x g for 1 hr, 2-ml aliquots of the cytosol fractions were applied on the column. Elution was carried out at 4' at a flow rate of 6 ml per hr were collected. with Buffer D. One-ml fractions Column, 2.5 x 30 cm; bed volume, 153 ml.-, lung cytosol; w liver cytosol;U , fetal serum (lo-fold dilution with Buffer D). 413

Vol. 47, No. 2, 1972

BIOCHEMICAL

RESULTS AND DISCUSSION. and lung

contain

to that

a cortisol-binding

G-ZOO.

and that by serum

greater

amount

to liver

It

Cellulose cytosol

which

indicate

that

tisol-binding

that

cortisol-binding the

(Fig.l),

the

lung

molecule

in addition

of the lung is

is not

a minor present

the rabbit

to possible

and liver

in

In addition

fetal

liver lung

examined

However,

appears

cells

to the major

a far

to contain

compared a cellular

by serum

CBG-like

(Fig.1).

components

CBG.

on DFAEcomponent,

with

the void These

one or more cytoplasmic of these

contami-

cytosol

(eluted

or in serum

contain

The specificity

since in

on

due to contami-

by chromatography

component

cytosol

blood

1. is

component

similar

by filtration

contain

in Fig.

liver

pattern

contamination

cytosols

cortisol-binding

macromolecules. yet been

at least

fetal

an elution

fractions.

cytosol

rabbit

cytosols

shown

to the CBG-like

in progress.

contains

has not

tissue

bound

with

and lung

component

in

from

(CBG) when

liver

is

columns

volume)

binding

likely

fractionation

cytosols

macromolecule

of cortisol

cytosol

that

globulin

CBG present

cortisol-binding Further

is

the

nation

lung

1 shows

of serum cortisol-binding

Sephadex nants

Figure

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

for

results cor-

cortisol-

studied.

50 r

(%I-Cortisol)

X 10m9

M in Incubation

Medium

of 3H-cortisol uptake by rabbit fetal lung nuclei. OneFig. 2. Kinetics gram aliquots of lung minces (from fetuses at day 28 of gestation) were incubated with increasing amounts of 3H-cortisol in 10 ml of Eagle's Hela tissue culture medium at 37O for 2 hrs. Purified nuclei were prepared as described in METHODS. The purified nuclear pellets were homogenized in Buffer B and aliquots of the homogenates were extracted with 3 ml of ethanol and counted. Separate aliquots of the purified nuclear pellet homogenates were taken for DNA determinations. 414

BIOCHEMICAL

Vol. 47, No. 2, 1972

The kinetics rabbit

fetal

binding

lung

minces

is

reached

sites

medium

of cortisol of 3H-cortisol

Table

1, the nuclear with

the ability

Table

at 37'

is shown

This

in Fig.

(probably uptake

of different

2.

cortisol

in

compete

glucocorticoids

in the

is within

observed

fetal for

further

is

specific

the binding

sites.

to compete

with

incubatron

the physiological

(Fig.2).

lung

of

of the nuclear

concentrations, is

of 3 H-cortisol

incubations

Saturation

concentration

non-specific)

activity

following

concentration

At higher

glucocorticoid

1.

of 3H-cortisol

when the 3 H-cortisol

in blood.

uptake

steroids

uptake

5 x 10 -8 to 1 x 10-7M.

is

range

of the nuclear

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

nuclear

As shown since

in

only

In addition,

3H-cortisol

uptake

by

EFFECT OF NON-LABELED STEROIDS ON THE NUCLEAR UPTAKE OF 3HCORTISOL IN RABBIT FETAL LUNG*

Non-labeled in incubation

Nuclear uptake of 3H-cortisol (cpm/ IJZSDNA)

steroid added medium

% Competition

43.5 Cortisol

6.8

84

9a-Fluorocortisol

4.1

91

Corticosterone

19.6

55

Estradiol-17@

39.6

9

Progesterone

44.5

0

Testosterone

45.8

0

* One-gram aliquots of rabbit lung minces (from fetuses at day 28 of gestation) were incubated in 10 ml of Eagle's Hela medium containing 1 x 10M7M 3H-cortisol with or without a ten-fold excess of non-labeled steroid for 2 uptake of 3H- cortisol was measured as described in hours at 37O. Nuclear Fig. 2. lung

nuclei

tritiated nuclear

steroid extract

Preliminary is

correlates

cortisol.

well

could 50-60%

evidence Fig.

with

their

be extracted of the

labeled

indicates 3 also

shows

that that

biological from

lung

steroid the

labeled

the

amount

415

activity. nuclei

About

with

is bound steroid of bound

Buffer

70-90% C.

In the

to macromolecules extracted steroid

from

of the

(Fig.3). the nuclei

in nuclear

ex-

Vol.

47, No.

2, 1972

BlOCHEMlCAL

AND

BIOPHYSICAL

RESEARCH

Rabbit Nuclear

*E

COMMUNICATIONS

Fetal Lung Extracts

3000

r! t I

2000

4 1000

4

a

16

12

Fraction

20

24

32

28

Number

Fig. 3. Gel filtration on Sephadex G-50 columns of rabbit fetal lung nuclear extracts prepared after incubation of lung minces (from fetuses at day 28 of gestation) with:-, 1 x IO-7M 3H-cortisol;+.0 , 1 x 10s7 3H-cortisol+ 1 x 10m6M estradiol-17B;H , 1 x 10e8M 3H-cortisol+l x 104M cortisol. Techniques of tissue incubation and preparation of nuclear extracts with Buffer C are described in METHODS. tracts

is

ten-fold

greatly excess

has no effect. rabbit

from

cortisol

for

of the

specific

cortisol

taken

gestation

newborn

a limited trations

but

incubated

a ten-fold

of the nuclear

experiments

lung

at 370 as these

nuclear

uptake

sites

and minimal expressed

earliest

studied)

time lower

tissues

excess

found

of a

of estradiol-17@ in

incubated

non-specific

reaching

the presence

and of newborn

were

as cpm/pg

in

of cortisol

were

conditions

up by nuclei,

Somewhat

lung

tissues

rabbits

is

with

in saturatio

The amount

DNA, increases

shown

1 x 10m7M 3H-

to result

uptake.

of

of 3H-

from day 20 of

a maximum at about

nuclear

uptake

of cortisol

was observed

in

report

demonstrate

that

28-30

days of

in lungs

of

rabbits.

The results nuclei

cortisol

is

day 20 to day 30 of gestation

2 hours

(the

gestation.

tissue

of non-labeled

In these

4.

when lung

A comparison

fetuses

in Fig.

decreased

contain number

presented

this

a macromolecule of binding

of cortisol,

with sites

Preliminary

a specific which

are

evidence

416

affinity

for

rabbit

fetal

glucocorticoids

saturated

with

physiological

indicates

that

cortisol-binding

lung and concencom-

BIOCHEMICAL

Vol. 47, No. 2, 1972

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Gestational

Age (days)

Fig. 4. Changes in uptake of 3H-cortisol (per unit DNA) by lung nuclei of the developing rabbit fetus. Each point represents the mean of 5-6 determinations. For each determination, lungs from all fetuses of the same litter (5-10 fetuses) were pooled, the tissue was minced and a one-gram aliquot of the minces was incubated in 10 ml of Eagle's Hela medium containing 1 x 10m7M 3H-cortisol for 2 hrs at 370. The nuclear uptake of 3H-cortisol was then measured as described in Fig. 2.

ponents

may also

interest

is

ses during This

the finding the last

observation

in lung

be present

extracts

the cytoplasm

the nuclear

of gestation

correlates

well

of rabbit

fetuses

and is

of fetal

uptake reaching

cells.

of cortisol

Of particular

in lung

a maximum at about

the concentration (10,14,15).

markedly

lung

tissue 28-30

of surface-active Pulmonary

increased

from

increadays.

phospholipids

surfactant

is

detectable

day 24 to the end of gesta-

(10,14,15). The physiological

significance

the present

time.

liver

is mediated

is

that

third

at day 24 of gestation tion

in

cells known

synthetase, adrenal

that

It

a variety tyrosine

steroids.

enzymes necessary

of these

has been proposed by cytoplasmic of enzymes

(e.g.

for

surfactant

is difficult

the mechanism

and nuclear

aminotransferase) One could

that

findings

that

synthesis

417

cortisol

induction

receptors

(11).

alkaline

can be induced

speculate

of enzyme

cortisol

invertase,

phosphatase,

in the induces

and stimulates

to evaluate

lung

immature

at in It

glutamine animal

by

one or more of the maturation.

Vol. 47, No. 2, 1972

ACKNOWLEDGEMENTS. excellent technical

BIOCHEMICAL

The authors assistance

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

would like to express their appreciation of Ms. A Dillon and Mr. R Linderi

for

the

REFERENCES. 1. Avery, M.E. and Mead, J., Am. J. Dis. Child 97, 517, 1959. 2. Clements, J.A., Physiologist 5, 11, 1962. 3. Abrams, M.E., J. Appl. Physiol. 21, 718, 1966. 4. Pattle, R.E. and Thomas, L.C., Nature 189, 844, 1961. 5. Buckingham, S. and Avery, M.E., Nature 193, 688, 1962. 6. Humphreys, P.Q. and Strang, L.B., J. Physiol.(London) 192, 53, 1967. 7. Buckingham, S., McNary, W.F., Sommers, S.C. and Rothschild, J., Federation Proc. 27, 328, 1968. a. Liggins, G.C., J. Endocrinol. 45, 515, 1969. 9. DeLemos, R.A., Shermeta, D.W., Knelson, J.H., Kotas, R.V. and Avery, M.E., Pediat. Res. 3, 505, 1969. 10. Kotas, R.V. and Avery, M.E., J. Appl. Physiol. 30, 358, 1971. 11. Rasps, G. (ed.) Schering Workshop on Steroid Hormone Receptors, Advances in the Biosciences 7, Pergamon Press, 1971. and N.O. Kaplan (eds.) Methods in Enzymology, 12. Schneider, W.C., in S.P. Colowick vol. 3, Academic Press, New York, 1957, p.680. 13. Giannopoulos, G. and Gorski, J., J. Biol. Chem. 246, 2530, 1971. 14. Kikkawa, Y., Motoyama, E.K. and Gluck, L., Am. J. Pathol. 52, 177, 1968. 15. Gluck, L., Sribney, M. and Kulovich, M.V., Pediat. Res. 1, 247, 1967.

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