Calmodulin modulates prolactin secretion in vitro: Studies with calmodulin containing liposomes

Life Sciences, Vol. 41, pp. 2437-2444 Printed in the U.S.A.

Permagon Journals

CALMODULIN MODULATES PROLACTIN SECRETION IN VITRO: STUDIES WITH CALMODULIN CONTAINING LIPOSOMES Gennaro Schettini, Tullio Florio, Olimpia Meucci, Elisa Landolfi, Michael J. Cronin* and Robert M. MacLeod** Department of Pharmacology, II School of Medicine, University of Naples, via S. Pansini 5, 80181 Naples, ITALY. Departments of *Physiology and ~ I n t e r n a l Medicine, University of Virginia, Charlottesville, Virginia 22908, U.S.A. (Received in final form October 2, 1987) Summary ++

The control of prolactin secretion by Ca +calmodulin and cyclic ++ AMP was studied. Ca ionophore A28187 stimulated both cyclic AMP accumulation and prolactin release by primary culture of anterior pituitary cells in vitro. The increase of cyclic AMP formation by A23187 preceded that of prolactin release. To test the calmodulin involvment in these processes we used either selective calmodulin antagonist, the naphtalene sulphonamide derivative WT, or calmodulin containing liposomes. W7 dose dependently inhibited both basal or A23187 stimulated cyclic ++ AMP accumulation and prolactin secretion. Insertion of Ca + calmodulin within the cells stimulated prolactin secretion without ++ modifying cyclic AMP accumulation. W7 inhibited the Ca +calmodulin containing liposomes stimulation of prolactin release. These results suggest that calmodulin partecipates to the process of prolactin release.

++

Many

regulatory

functions

of

Ca

++

are

mediated

through

the

Ca

binding

++

receptor protein, calmodulin (CDR) (1,2). The binding of Ca to CDR has been suggested as an important step in regulating secretory+ processes in various cells (1,3). In addition CDR is necessary for the Ca mediated stimulation of enzyme activities, such as adenylate cyclase in the brain (4), pancreatic ++ islets (5), adrenal medulla (6), pituitary gland (7,8). Ca and cyclic AMP (cAMP) are intracellular messangers which mediate stimulus-secretion coupling ++ in eukariotic cells. Some secretagougues preferentially alter Ca metabolism to initiate a secretory response, with a subsequent modification in cAMP metabolism; however the reverse sequence of events has been reported (3,1). Both second messangers are involved in the control of prolactin (PRL) release from anterior pituitary cells in vitro (iO,11). In the present study we test the involvment of CDR in regulating PRL secretion either by using a CDR antagonist, the N-(6-aminohexil)-chloro-l-naphtalenesulphonamide derivative W7 (12,13), or by inserting CDR within anterior pitui0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.

2438

Calmodulin

tary to

cells

by means

antagonize

into

the

chinese

level

CDR

Indeed within

CDR

allowed

us

are

biological

the

cell

liposomes. ovarian

to

liposomes

effective

of

of PRL S e c r e t i o n

The

CDR-dependent

hamster

similarly

taining

Modulation

CDR

itself

also

The

use

(12).

artificial

compounds

otherwise

liposomal

subcellular

preparations

proven

to

substances,

impermeable

22, 1987

incorporation at

how CDR affects

vescicles

of m a c r o m o l e c u l a r

thymidine

distribute

of

elucidate

lipid

H

to

41, No.

W7 has been demonstrated

and

and

to more directly

carriers

antagonis~

proliferation cells,

Vol°

to

the

be

con-

PRL release. versatile

able

cell

to

and

transport

membrane

(14).

Methods

Pituitary in

cell

monolayer

cells

were

culture: culture

allowed

at

controlled

atmosphere. preincubated

For At

stored

(IBMX)

of

at -20°C

ethanolamine to

the

was

added

the

lipids was

aliquots soaked

the 0.i

medium

N

HCI

Unilamellar

described in

the

test

substances

cells

were

not

was

for

quickly

i0

at

and replaced

for

15

preincubated

removed

minutes

vulume

micelles. in

placed To

the

liposomes Briefly,

amount

two in

the

Then

for

37°C.

2

were

parts.

membrane

I0 mg/ml stream,

was

buffer

to eliminate

first

dialysis

part

were washed

octylglucoside

spinning The

obtained

PRL

All

minutes.

with IBMX. assay,

samples

by detergent

phosphatidylserine

of p h o s p h o s a l i n e

After

second

of

under N

chloroform.

desired

divided

(ig).

vortexing

residual

PBS.

and the

was then removed

and were

till assayed.

formed

and in

with

(Avantl)

with

IBMX

liposomes

study,

up g~n~ly

eliminate

tion

with

preparation:

dried

The

in a humidified

The medium

containing

twice with serum

kept

activity.

sis as previously were

to the plates

and

al.(ll).

phosphodiesterase

extracted

Liposomal

et

(Gibco)

medium

They were washed

prepared

free R P M I - 1 6 4 0

end

was

days to attach

were

Schettini

with 0.2 mM isobutylmethylxantine

experiments

the

cAMP

three

cells by

two hours

fresh the

pituitary described

for

and

with

anterior

previously

least

medium

to inhibit

Rat

as

part

was

Spectrafore

added

CDR

in

used.

The

phospholipids

twice with

diethylether

(Calbiochem) (PBS)

and

the foam, further 3.5 K the

dialy-

and phosphatidyl-

(60 mg/ml)

vortexed

divided

(18mm)

amount

till

the preparain

two

previously of

i mg/ml,

then it was divided in two aliquots and placed in membrane dialysis as above. The four aliquots were dialysed for three days, changing the buffer after 16

hours

and

then

every

24

hours.

Two

dialysing

buffers

were

prepared:

PBS

with or without CaCI lOOuM in order to obtaine the following samples of lipo2 somes to test: sample i, liposomes alone, dialysed only against PBS; sample ++ 2, liposomes with Ca , dialysed against PBS + CaCI2; sample 3, liposomes++ with CDR added, dialysed against PBS alone; sample 4, liposomes with Ca and CDR, dialysed against PBS + CaCI . Two samples of the second aliquots 2 of liposomes were added with SIO0 or parvalbumin (PVA) (img/iml) respectively instead of CDR and dialysed against PBS + CaCI . The six liposomal preparations 2 were incubated for 2 hours with primary culture of anterior pituitary cells to Lest their effect on PRL secretion. To check the interaction of liposomes with cells, the fluorescent dye 6 - c a r b o x y f l u o r e s c e i n (Eastman) was used (16). The relative uniform distribution most of the dye has been released

of fluorescence in the cells suggests that within the cytoplasm following cells liposo-

Vol.

41, No.

22, 1987

mes fusion.

The percentage

Radioimmunoassay: and

protocols

Results

were

system

maximal

did

not

provided

Center

Test substances: The

in

by

terms

were measured

(17),

and Training

provided

expressed

varied

with a double

the of

National

NIADDK

according by

cells

of PRL S e c r e t i o n

the

from 40 to 60%.

antibody

Hormone

rat

PRL of

RIA using materials

and

RP-2

to the procedures

University

2439

Pituitary

standard.

Program.

Cyclic

of Gammaflow

Virginia

AMP

automated

Diabetes

Research

RIA core laboratory.

W5 and W7 (Caabco),

concentration

affect

Modulation

oF fluorescent

PRL was determined

kindly

concentrations RIA

Calmodulin

cAMP

of

formation

A23187

ethanol or

were dissolved

(1%)

PRL

at

which

secretion.

in absolute

the

IBMX

cells

was

ethanol.

were

dissolved

exposed directly

in RPMI 1640 medium. All agents were freshly prepared. CDR, SIO0 and PVA were a generous gift of Dr. Kretsinger, Dept of Biology, University of Virginia. All other reagents Statistics: dishes.

Assays

Data

from SIGMA

were

were

if not otherwise

performed

expressed

Statistical

once.

were

in duplicate

as mean

significance

+ SEM;

specified.

on samples

experiments

was determined

from

were

quadruplicates

repeated

at least

using ANOVA.

Results

++ Ca

ionophore

effect

of

ionophore content

A23187

A23187

on

(+200%; of

greater

cAMP

than

by A23187

until

Addition

of

At

3,

7,

than

at

l.B

control

values.

3 min

(p(O.05)

W7

to

release

till

was

of W7, 65%

preparations

we

60

min,

release.

exposure the

(p(O.Ol) (data

The

to the

intraeellular but was

significantly

always

stimulated

(Fig i). not

presented)

and

(Fig 2).

had

a less

reduction tested

PRL

of

to p~O.Ol)

basal

and PRL release

and

min

not

at 30 min

inhibited

analogue

liposomal

1.5

(p(O.05

produced

different

less

30,

min

and peaked cells

cAMP accumulation a

15,

accumulation after

active

six

WB,

pituitary

PRL

cAMP

peaked

both parameters. At lOOuM, W7 reduction in cAMP accumulation. The

contrast

less

both

formation

p(O.Ol). was

A23187-stimulated In

stimulated cAMP

were

++

alone, liposomes + Ca , liposomes + CDR, liposomes PVA. Aliquots (3,10,30,5Oui) of each preparation of

powerful

in PRL

effect

release

divided

in:

and

on 90%

liposomes

++

+ Ca + CDR or SIO0 or liposomes were added and

incubated

for 2 hours with primary cultures of anterior pituitary cells. Lipo++ somes prepared in absence of added Ca , containing only phospholipids (liposomes alone), or phospholipids + CDR (liposomes + CDR), and liposomes containing ++ phospholipids + Ca (liposomes + Ca ++ ) did not significantly stimulated PRL ++ release, while 30 and 50ul of liposomes containing Ca + CDR significantly enhanced PRL secretion (p
2440

Calmodulin

Modulation

of PRL Secretion

2

30 25

-

"6 ° E CL

Vol.

41, No.

22, 1987

1200 1000

I

20

_~F--

--

--

~ 800

15 10

,),

~ /~ 2OO

.53

7

15

30

60

.53

7

15

60

MINUTES FIG i Time

course

and

PRL

of

the

release

by

effect

of

primary

i0

Each

value

represents

the

mean

Left

panel

represents

the

control

the cells

exposed

A

,7, -= 50 2~

0

,

-6

A23187

on cAMP

of anterior

+ SEM

of

four

values.

accumulation

pituitary

samples

Right

panel

cells.

per

group.

represents

to lOuM of A23187.

125[ ~

'~

uM

culture

-5 log M W 5 or W7

~ 125

~

50.

=.

25

-4

-6 log M W 5 or W 7

FTG 2

Effect

of W5 and

W7

on A 2 3 1 8 7 - s ~ i m u l a t e d

cAMP accumulation

A) and PRL release (panel B) by primary cultures of pituitary cells. W5 did not affect A 2 3 1 8 7 - s t i m u l a t e d cAMP lation or PRL release, w7 significantly inhib-'zed (o{0.01) stimulabed cAMP accumulation at 5, i0, 50 and i00 uM release at i0 (p(O.05), 50 and i00 uM (p40.Ol).

(panel

anterior accumuA23187and PRL

_m >" ~

Vol.

41, No.

22, 1987

C a l m o d u l i n M o d u l a t i o n of PRL S e c r e t i o n

2441

TABLE I ++

E f f e c t of l i p o s o m a l p r e p a r a t i o n s c o n t a i n i n g Ca

++

, CDR, or Ca

+ CDR on

PRL s e c r e t i o n by p r i m a r y c u l t u r e of a n t e r i o r p i t u i t a r y cells in vitro. a m o u n t of l i p o s o m e s

RIA PRL n g / w e l l 3

10

30

50

l i p o s o m e s a l o n e+ +

3 0 4 +. 9

3 5 0 +. 2 0

345+12

356+16

liposomes

+ Ca

278+6

338+19

375+14

liposomes

+ CDR + +

2 9 6 +. 7

liposomes

+ Ca + +

+ CDR

liposomes

+ Ca + +

liposomes

+ Ca

Each value posed

to

liposomes with

was

(ul)

alone

liposomes

3 0 2 +. 1 7

3 4 8 +. 1 0

.

3 6 5 +. 1 6

.

+ PVA

3 1 5 +. 8

.

3 4 8 +. 1 4

.

+ $100

333+13

the for

treated

hours,

group.

effective

.

351+18

mean + SEM o f

two

was 2 8 4 + 1 2

significantly

.

305+14 .

represents liposomes

.

6 wells

*p(O.05;

The a v e r a g e

~p

per

346+14

348+18

570+24**

429+14 ~

341+9

337+6

337+9

345+17

group,

<0.01

PRL v a l u e

Cells

versus of

cells

were

ex-

respective not

treated

ng/well.

only

at

the h i g h e r

concentration used

(TABLE

II).

CDR d i r e c t l y a d d e d to cell c u l t u r e did not m o d i f i e d PRL secretion.

T A B L E II

E f f e c t of W5 and W7 on the s t i m u l a t i o n of PRL s e c r e t i o n by 30ul l i p o s o m e s ++ c o n t a i n i n g Ca + CDR. RIA PRL n g / w e l l control

liposomes

253+9

++

l i p o s o m e s + Ca

+ CDR

356±17§

++

W7 i0 uN + l i p o s o m e s + Ca

+ CDR

354+10

+ CDR

269+18 ~

++

W7 30 uN + l i p o s o m e s + Ca ++

W7 60 u M + l i p o s o m e s + Ca

+ CDR

200+11 *~

++

W7 i00 uM + l i p o s o m e s + Ca

+ CDR

2 4 4 + 1 2 *~

++

W5 30 uM + l i p o s o m e s + Ca

+ CDR

314+17

+ CDR

310+20

++

W5 60 uM + l i p o s o m e s + Ca ++

W5 i00 uN + l i p o s o m e s + Ca

+ CDR

252+23 ~

E a c h v a l u e r e p r e s e n t s the mean ! SEN of 6 wells per group.

Cells were p r e -

t r e a t e d for 30 min w i t h W5 or W7 and then, s i m u l t a n e o u s l y ,

e x p o s e d to W5

or W7 + Ca ++ + CDR + l i p o s o m e s for 2 hours. CDR c o n t a i n i n g l i p o s o m e s t r e a t e d cells.

*p40.05;

*~p~O.Ol versus

Ca++ +

§p O.O1 v e r s u s c o n t r o l l i p o s o m e s

2442

Calmodulin Modulation

of PRL S e c r e t i o n

Vol.

41, No.

22,

1987

Discussion

++ Ca

and

cAMP

activities (ii)

in

been

proposed

PRL

are

(3).

the

release

and

often

several

control

of

PRL

(ii).

We

showed

from

significantly release,

integral

Although

secretion

pituitary

stimulates

suggesting

action

modulation

of the s t i m u l u s - s e c r e t i o n

Calmodulin

has been r e c e n t l y

tion

(11,18,19).

derivative

W7

In

(12,13),

cAMP a c c u m u l a t i o n The

inhibition

effect

on

our

of

cAMP

et al

(13),

similar

diesterase and

PRL

dent

PRL

between

to

(PDE)

the

(13)

and

W7 as

to

also

of m a g n i t u d e

higher

compared

sensitivity

cannot

could

possible

to

test

the plates. In contrast to

CDR

was

and

less

Thus

the

since less

to

were

unable

for

W7

showed

insertion

PRL

an

CDR

to

inhibit

the

present

to

inhibit

PRL secre-

A23187

PRL

PRL

3

stimulated

from

the

W7

sensitivity

activated

study,

action.

WS,

release basal

and

in PRL

by

show

activities.

affecting

this

has

the

also

not

been

cells

from

3 d i s p l a c e d Hkr7 binding

240uM

respectively

concentration stimulated

The

results

cellular

detach

which

liposomes

lacto~rophs.

almost one order

of W7,

and

in the

formation ++ Ca depen-

These

but

phospho-

cAMP

phospholipid

secretion,

of 210uM

of

than

HW7 from CDR was 31uM

CDR

concentrations

IC50

involvment

marked

among different

antagonist

range

cAMP

we

we

(13), used.

formation

and

p r e p a r e d liposomes ++ Ca + CDR was

containing

other

liposomal

preparations

stimulate PRL release. Thus CDR inserted within ++ in Ca bound form, stimulates PRL release, s u g g e s t i n g

in the control

experiment

IBMX,

thus

the

Hence

the

This

in the

of

formation

and

cells

by

apparent

performed

PDE

of PRL secretory

cAMP

of basal

within

formation.

cells.

the

sulphonamide

different

to displace

concentratios

W7

preparation

modulation

that

the

PRL

enzyme and the PRL secretory

inhibiting

reduce

inhibit

CDR

release

with in

to

the

inhibition

of

A23187

significantly

CDR

nucleotide

CDR

with

a

secretion

the

via liposomes,

as

and

but for c o n c e n t r a t i o n s

higher

CDR

PDE

W7

that CDR is involved As

reported

higher

to reduce

only

to

that

regulating

less

to

address

enhace

the cells

W7 in

(20,21)

was

due

activity.

CDR;

able

such

W7

is likely due to its CDR i n h i b i t i n g

directly

containing

and

showed

activities.

required

to the

potent

of

for

inhibit,

that

activated

ability

IC50

effectively

effective

PRL release To

the CDR

has

accumulation

naphtalene

basal

cyclase

to the W7 effect

exclude

more

by

likely

cellular

been

(C-kinase)

we

inhibited

is

concentration

kinase

C-kinase

we

antagonist

the a d e n y l a t e

that

the

release.

However

CDR

produced

This

of various

reported

different

Here

in the m e c h a n i s m s

the

secretion

protein

the

cAMP

and cAMP role

c o u p l i n g of PRL.

dependently

accumulation.

the r e s u l t a n t

Hidaka

(I0)

a complementary

before the ionophore enhances +÷ and cAMP are i n t e g r a t e d in

Ca

implicated

study,

dose

(ii).

many cellular

Ca

and PRL release.

to the CDR blockade, process,

of

for

stimulates

vitro

accumulation

the

roles

recently

A23187

in

in c o n t r o l l i n g

suggest

only

that

cells

cAMP

that

interactive

studies

could

changes

in

stimulated means

of

liposomes

activated

cellular

cAMP

process.

concerned, cAMP

by

may the the

the

studies

accumulation,

liposomes

discrepancy

with be

is

be

did

not

explained

cells

were

insertion

content

could

with

while

modified by

the

the the fact

not p r e t r e a t e d ++ Ca ~ CDR

of be

difficult

to

detect if both adenylate cyclase and PDE a c t i v i t i e s were increased c o n c u r r e n t l y ++ by Ca + CDR. Moreover, recently has been r e p o r t e d that in the membrane prepa-

Vol.

41, No. 22, 1987

Calmodulin Modulation

of PRL Secretion

2443

rations of anterior pituitary glands, adenylate cyclase activity was stimulated ++ by Ca and CDR. Thus reduction of cAMP formation induced by W? may likely partecipate to the W7 inhibition of PRL secretion, being cAMP one of the component involved

in the control of PRL secretory

process.

In conclusion these results show that CDR partecipates in the mechanisms gover++ ning PRL release and that the involvment o f Ca in the stimulus - secretion coupling

of

an activation

PRL

appears

to

be

mediated

of cAMP generating

system

via

CDR,

and may be partially

due

to

in the lactotrophs.

Acknowledgement

We and of

gratefully

acknowledge

G.Meyers. CDR,

SIO0

Virginia 07535

for

the technical

We

also

wish

to

and

PVA,

and

the

assaying

(R.M.M.);

cAMP.

NS 18409

thank

assistance

Dr

Diabetes

This

Core

research

(M.J.C.);

of C.Valdenegro,

R.H.Kretsinger Laboratory

was

1 F05 TWO

gift

of the University

supported

3267

M.MacOeen

for the generous by

(G.S.);

USPHS

Grant

CNR cont.

of CA-

860062404

(G.S.).

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i.

W.J.

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A.R. MEANS and J.R.

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M.W.

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21.

Calmodulin Modulation of PRL Secretion

Vol. 41, No. 22, 1987

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