Early effect of steroids on 45calcium uptake by mouse thymocytes

Vol.102,No.1,1981 September

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 458-465

16,1981

EARLY EFFECT OF STEROIDS ON 45CALCIUM UPTAKE BY MOUSE THYMOCYTES F. Homo and J.

Simon

INSERM U7, Physiology and Pharmacology, 161 rue de Sivres, 75730 Paris

Received

H8pital Necker, Cedex 15

May 19,1981

SUMMARY : The effects of various steroids on 45calcium uptake wereinvestigated in parallel with the viability of mouse thymocytes. Dexamethasone, a synthetic compound with glucocorticoid activity, induced a rapid increase in membrane permeability to calcium. This effect was still measurable using 1O-7 M dexamethasone and appeared specific for compounds with glucocorticoid potency. In addition, calcium efflux from prelabeled cells was not altered in the presence of dexamethasone, indicating an increased total cell concentration. It is therefore suggested that calcium ions play a role in steroid-induced cell lysis.

Although cations

alterations

have been reported

steroids

in several

available

on the

tration

of

diol-17

8, even

cellular

exchange of

grown

the

scanty, of

the

effect

(1).

(2).

that

ionophore evidence

on 45calcium

presented uptake

1 hr (3)

play

cell for

a rapid

by isolated

0 I98I by Academic Press, Inc. of reproduction in any form reserved.

vitro

thymocytes.

458

calcium

In

in

(5)

effect

data the

are

action

has been tissue,

and the

the

calcium

as well In

fully

transport

metabolism lymphoid

(6,7j.

produce

direct

steroid-induced

lysis in

0006-231X/81/170458-08$01.00/0 Copyright All rights

(4).

in

affect

ion

documented

a role

levels

of

in vitro to

can influence

calcium

are well

to

estrarates

observed

calcium

is

administhat

influences

Although

glucocorticoids

A23187-induced is

similarly

and Tenenhouse

after shown

as 2.5 min after

free

with

information

shortly

has been

intracellular

divalent

treatment

little

has been

as early

intracellular

of glucocorticoids suggested

cells

mucosa within

by Rasmussen

been

It

other

in vivo

of calcium

cells.

Glucocorticoids

possibility

term

tissues,

Progesterone

in

and

concentrations,

in uterine

AMP through

suggested has

isolated

at physiological

intestinal

cyclic

long

exchange

to

increase

oocytes

across

metabolism

following

cellular

hormone

rapid

calcium

hormone-responsive

steroids

addition very

in

this

ion as in

paper,

of glucocorticoids

Vol. 102, No. 1,198l

BIOCHEMICAL

AND

MATERIALS

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

AND METHODS

Animals and reagents The animals used in all investigations were intact female C57B16 mice, 6-8 weeks old. Unlabeled steroids were obtained from Sigma Chemical Corp. (St. Louis, MO). Each steroid was dissolved in absolute ethanol and diluted to the appropriate concentrations before each experiment. The final ethanol concentration was always lower of equal to 0.01 % and did not alter 45calcium uptake or cell viability. Calcium concentrations were adjusted using a calcium standard solution (Titrisol, Merck). EGTA (ethylene glycol-bis (B-aminoethylether) NN'-tetra acetic acid) was purchased from Sigma. Culture medium (minimum essential medium, MEM, calcium free, ref 138) and the various supplements (sodium pyruvate, L-glutamine, penicillinstreptomycin and non essential amino acid solution) were obtained from GIBCO. Sterile 45calcium chloride solution (20-25 Ci/g) was purchased from CEA (Saclay, France). Isolation of thymocytes The procedure for thymocyte isolation has been previously described in detail (7,8). Cell suspensions were adjusted to contain lo7 cells/ml in MEM supplemented with sodium pyruvate (ImM), L-glutamine (2mM), 1 % (v/v) non essential amino acid solution, 100 U/mL penicillin and 100 ug/ml streptomycin and usually 1 mM Ca++ (medium A). Cell preparation was carried out at room temperature and the cell suspensions were preincubated at 37-C for 30 min in an atmosphere of 5 % CO2 in air before each experiment (9.). Measurement of calcium uptake At the beginning of the experiments, aliquots of the cell suspension were added to glass tubes containing 45calcium (1 uCi/ lo7 cells) and either steroid or vehicle only (control) in a final volume of 4 ml. After various periods of incubation at 37-C, 100 ul aliquots of the cell suspensions were rapidly filtered on Whatman GF/A filters. The filters were washed with 2 x 10 ml of ice cold buffer (135 mM NaCl, 5 mM KH2 P04, 5 mM Tris-HCl, 1 mM MgC12, 5 mM The radioactivity collected on the filters was glucose, pH 7.4). counted by liquid scintillation spectrometry. In the experiments designed to folJ!w calcium efflux, $11 suspensions were incubated first with calcium (1 uCi/lO cells) for 2 h at 37-C, in order to allow tracer equilibration. Cells were then rapidly washed twice by centrifugation (Eppendorf 3200 centrifuge, 11,000 g, 15 set) and resuspended in medium A supplemented or not with steroid or EGTA (time zero of the efflux). At various intervals following resuspension, Sam les were filtered as described above. At each time the amount of 85 Ca associated with the cells was expressed as a percentage of the total bound calcium at the moment of steroid addition (time zero). Oetermination Viability sion technique tion procedure.

of cell viability of the cells was estimated bv the trvoan blue (10) and was always over 95 %-at the end of the

459

excluisola-

BIOCHEMICAL

Vol. 102, No. 1,1981

Ifi

0 515

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS

1

1

1

1

I

30

60

120

180

24(

TIME

jminutet)

Figure 1 : Dose-response curves of the action of dexamethasone on g5calcium uptake. The concentration of dexamethasone tested were 10-8M ( 0 ), lo-7 ( LI ), 10S6M ( 0 ). Each value is expressed as a percenand represents the mean tage of the control sample (100 % : control) (+ S.D) of triplicate determination in 6 experiments.

RESULTS As previously 45calcium after

described,

increased the

glucocorticoid, diene-3,20-dione)

to

crease

in calcium

uptake

occured

as early

as 5 min

gradual

decline

in

ma? groups riments

were

with

of

the

the

lo-6M

17,21

of

time

drugs to

the

uptake

reach

a

cell

dexamethasone

of

plateau

suspension

after

rate

a rapid (Figure

steroid of

(a

synthetic

,trihydroxy-16a-methylpregna-l-4

was demonstrated

as reported

and Edelman (12), kinetics of calcium

absence

(7,9). addition

9 fluoro-118,

be emphasized,

the

progressively

60 min incubation Following

in

addition

45ca?cium

by Jensen

and 1).

transient This

was followed

accumulation.

and Rasmussen

(11)

in-

peak which by a

It

should

and

Kaiser

that a large variability was observed in the uptake and response to drugs among different ani-

on different qualitatively

days.

Although

similar,

both

460

the

curves

from

the magnitude

several of the

experespon-

BIOCHEMICAL

Vol. 102, No. 1,198l

AND

I 10

0

I 20

BIOPHYSICAL

I 30

1 40

I 50

RESEARCH

COMMUNICATIONS

I 60

TIMt (nin)

Figure

2 : Effect The

then

rapidly

sion

in

were

filtered

45Ca

the

mean

to

shown

tectable

As of

was

in

the

1,

the

concentration

in

Figure

as

a typical

kinetic

this

time

and

resuspenaliquots the

a

amount

of

percentage

of

Each

is

resuspension.

of

and the

37-C

value

experiment.

experiment

( 0

was

)

repeated

results.

length

Therefore,

at

following

each

This

similar

2 h

dexamethasone,

expressed of

dexamethasone.

and

for

time

required

to

experiments

specificity

stimulation

were

of this of

obtain always

effect.

‘%alcium

of dexamethasone

the used

As al-

uptake

and was still

was de-

lo-7M.

shown

modify

tion

At

moment

qualitatively

Figure

upon at

10S6M

filters.

the

efflux.

45Ca

intervals

not

cells

at

dose-response

in

dependent

not

the

10m6M

variable. the

various

determination

)

gave

with

or GF/A

calcium

A

and

were

At

triplicate

(

study

twice.

whatman

dexamethasone

peak

incubated

with

of

3 times

so

on

on 45calcium

first

A containing

bound

control,

to

washed

associated total

dexamethasone

were

medium

the

se

of

cells

45calcium

the

calcium

2,

efflux chelator

addition from

of

10m6M

prelabeled

dexamethasone

cells,

EGTA enhanced

the

whereas

efflux

did addi-

(results

not

shown). studied

In order to explore the specificity the effects of various steroids

At

lo-6M,

so

enhanced

gesterone trogenic

other the

glucocorticoids uptake

of

(corticosterone 45calcium, a non

and diethylstilbestrol, activity

did

not

of this phenomenon, we have on calcium uptake (Table I).

stimulate

calcium

461

and

cortisol)

whereas

testosterone,

steroidal

compound

uptake.

alpro-

with

es-

I

isolated

71

3-6

of

+ +

116 111.4

113.3 t

37.2 31.4

2i.6

129.6 +

111.82

OIETHYLSTILBESTROL

PROGESTERONE

52.5

38.3

27.7

+ la4

various Each

value

(10w6M)

+

103.5 +

118.4

117.6 +

13

13

10.4

323.1 + 167.2

+

83.6 +

92.1

79.5 +

uptake

68.9

13.8

19.7

10.9

90’

67.8

89.9

15.6

25.3

99.9 + 108. a +

17.3

76.4 +

261.6 + 101.6

200.7 t

227.4 +

a percentage

triplicate

45Ca

al.5

of

351.6 + 137.2

243.6 +

60’

as

on SD)

254.8 + 116. a

expressed

mean(+

244.9 +

30’

is

the

TIME

% : control).

and

is

steroids

217.8 -+ 55.3

(100

experiments

233.9 + 107.6

239.9 +

15'

incorporation

from

+

115.2

TESTOSTERONE

37.7

55

Effects thymocytes.

315.7

293.3

CORTISOL

5’

control

:

+ 101.9

215.8 +

CORTICOSTERONE

+

226.9

OEXAMETHASONE

of

determinations

by

Table

+ 39.3

122.4 2 15.6

98.2 + 37

96.8

259.6 + 93.7

213.7 + 66.4

197.1 + 40.2

183’

Voi.102,No.1,1981

BIOCHEMICAL

Table in

II the

: Determination absence

cells

was

or

of presence

determined

AND

BIOPHYSICAL

cell

viability

of

using

10S6

trypan

RESEARCH COMMUNICATIONS

after

steroids.

blue

4 hr The

exclusion

of

incubation

number

of

Cell

viability

Control

90.2

% + 3.6

n = 13

Dexamethasone

70.7

% + 19.6

n=5

Corticosterone

81.6

% + 10.2

n=5

Cortisol

81.4

% + 4.2

n=5

Testosterone

91.6

% + 1.5

n=3

Diethylstilbestrol

91.2

% + 2.1

n=3

Progesterone

88.5 x54.8

In

parallel

experiments,

cells

after

cline

when glucocorticoid

n=3

we showed that the with the various drugs

4h of incubation

compounds

were

viable

procedure.

present

viability of the only began to de-

II).

(Table

DISCUSSION Calcium mical

ions

play

and biological

a major

be intimately

involved

in the

of cell

(15,16).

Despite

ning

lysis

the

early

interaction

metabolic

little

is

suggested ponsible cytoplasmic

in the modulation

of mammalian control all

effects

by Kaiser

on those

triggered events

and Edelman

of thymocytolysis activity.

cells

of cell the

with by the

linked (6)

that

and appear (14)

information their

hormone to

of many bioche(13)

proliferation

available

of glucocorticoids

known

Ca++

role

processes

cell

in

one of the

463

this

explanation

and the

lymphoid

It

cells, has

mechanisms

may be a glucocorticoid-induced However,

and

concer-

receptors death.

to

been res-

change was

discar-

in

Vol.102,No.1,1981

BIOCHEMICAL

ded because lymphoid

of the

cells

described

in

with

the

of any early

the

presence

A23187

(7).

We

on

45calcium

corticoids

rapidly

similar

to that

(7,ll).

In the

tributed

described

brane

followed

in

of

lymphoid

ionophore

increase

by

lysis

promoted

the

after

of

the

which

in-

glucocorthat in

gl ucoa manner

ionophore pattern

permeability

a redistribution

recently

of

45calcium

biphasic

by

by ionophore

effect

of

cells

this

in

of cell

demonstrate

uptake

uptake

We have

uptake

results

the

ion

(17).

the

Our

stimulate

to a rapid

extent

reinvestigated uptake.

case

the

COMMUNICATIONS

in calcium

steroids

of calcium

thus

RESEARCH

changes

of

between

stimulation

ticoids

BIOPHYSICAL

lack

a relationship

creases

AND

treatment has been

at-

plasma

mem-

of the intracellular

calcium

in thymocytes

appears

at

10w6M

not

In contrast

with

(11). This

effect

specific

for

alter

tracer

thasone, with

thus

cell

calcium that This

been

the

absorption

the

event

death can also of

to study

action

of

in

the

calcium link

be observed (17).

between

Further cellular

that

steroid

the ad-

death. it

fragility,

unequivocally

corticosensitive

shown

because

nuclear

by dexamePreliminary

2 h after

of cell

importance

increase

biochemical cell

concentrations

and steroid

signs

siup-

may be associated have

elevated

first

calcium

concentration.

spectrometry

is of extreme

first

now in progress

the

did

modified

action

calcium

was significantly

that

was not

glucocorticoid

before

corticoid-induced increasing

that

content long

steroids

cells

is

observation

presents

preloaded

atomic

demonstrated

cell

from

of intracellular

using

uptake

permeability.

suggesting

an elevation

dition,

as sex

calcium

efflux

experiments

on calcium

glucocorticoids

gnificantly take,

of steroids

linked in the

has already which to

gluco-

presence

experiments changes

reof are

in calcium

and corticoresistant

lymphoid

populations. ACKNOWLEDGEMENT

The authors are grateful to Dr Dominique Duval for his continued support and advice and to Dr Marie-Aude Devynck for helpful discussions. REFERENCES

1. Pietras, 2. Wasserman, L.D.

R.J.

& Szego,

W. J.,

Pinto,

Proc.Natl.Acad.Sci.

C.M, L.H.,

(1975) Nature 253, O'Connor,

USA 77,

464

357-359.

C.M. & Smith,

1534-1536.

L.D.

(1980)

Vol.102,No.1,1981

3.

BIOCHEMICAL

Harrisson,

H.E.

AND

BIOPHYSICAL

& Harrisson,

H.C.

RESEARCH

(1960)

COMMUNICATIONS

Amer.J.Physiol.

199,

265-271. 4. Rasmussen, H. & Tenenhouse, 1364- 1370. 5. Munck, of

A. & Leung,

steroid

Marcel

K. (1977)

hormones

Oekker,

A. (1968)

in Receptors

(J.R.

Pasqualini

6. Kaiser,

N. & Edelman,

I.S.

Ourant,

S.,

& Ouval,

Corn. 93,

385-391. 13,

9. Borle, O'Malley Jensen,

F.

II,

Part

(1978) Endocrinology

of action pp 311-397,

103,

936-942.

(1980) O.Biochem.Biophys.Res.

0.

Hatzfeld,

J. & Evrard,

in Methods

in enzymology

D.,

(1975)

ed.) M.C.

and mechanism

ed.)

C. (1980)

J.Steroid.

135-143.

A.B.

10. Raff, 11.

Homo,

Ouval,

Biochem.

USA 2,

New York.

7.

8. Homo, F.,

Proc.Natl.Acad.Sci.

Vol

(1971)

49,

pp 513-573,

Transpl.Rev.

Academic a,

(J.G. Hardman Press.

52-80.

H. (1977) Biochim.Biophys.Acta

P. & Rasmussen,

& B.W.

468,

146-

156. 12.

Kaiser,

N. & Edelman,

I.S.

(1977)

Proc.Natl.Acad.Sci.

USA 3,

638-642. 13. 14.

Rasmussen,

H. (1970) Science

Whitfield,

J.F.,

dale, 15. 16.

S.K.,

ber,

(1975)

J.L.

Schanne, Science

17.

F.A.X., 2&,

Nicholson, lJ,

MacManus,

T. & Swierenga,

El Mofty,

170, J.P.,

S. (1976)

Scrutton,

M.C.,

Amer.J.Pathol. Kane,

404-412.

A.B.,

Rixon,

R.H.,

In vitro

12,

Serroni,

A.,

79,

579-595.

Young,

E.E.

Boynton,

A.L.,

You-

l-18. Nicolini,

& Farber,

C. & FarJ.L.

(1979)

700-702. M.L.

& Young,

D.A.

165-174.

465

(1979), J.Supramolecular

Struct.