Regulation of cyclic AMP and cyclic GMP levels by adrenocorticotropic hormone in cultured neurons

Vol.

133,

No. 1, 1985

November

27,

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1985

Pages

286-292

REGULATION OF CYCLIC AMP AND CYCLIC GMP LEVELS BY ADRENOCORTICOTROPIC HORMONE IN CULTURED NEURONS Patrick

Anglard,

Jean

Centre

Received

October

Guy

Zwiller,

Vincendon

and

Jean-Claude

de Neurochimie du CNRS, 5 rue Blaise 67084 STRASBOURG Cedex, France 23,

Louis

Pascal

1985

SUMMARY. The effect of adrenocorticotropic hormone (ACTH) on the intracellular concentration of cyclic nucleotides was studied in cultures of neurons from embryonic chick cerebral hemispheres. Incubation of neurons with ACTH(l-24) in the presence of phosphodiesterase inhibitor isobutylincrease in cyclic AMP while methylxanthine resulted in a sustained rise in cyclic GMP level was transient. The values obtained for halfmaximal stimulation were 0.5 lo M and 0.03 nM for cyclic AMP and cyclic ACTH( l-24) stimulated guanylate cyclase GMP respectively. Concomitantly, activity (half-maximal stimulation at 0.02nM). These results suggest the existence of two distinct populations of ACTH receptors in neurons and provide the first evidence that cyclic GMP does mediate the action 0 1985 Academic Press, Inc. of ACTH in neurons. It is

is

taken

produced

and

affecting

both

phorylation tion

of

of ACTH

on

In

spite

and

membrane

of

been

proposed

tissue

metabolism

in

been

cyclic

neurons studies,

on

nerve

cells

recently,

(4)

cultured

rat

cortical

the

present

ACTH

AMP

metabolism,

to

for

is

was

to

the

be

in

phos-

neurotropic

effects

to promote glucose

of ACTH

action

effect

of ACTH

survi-

uptake

(5).

mechanism

in

of

Cyclic

AMP

rat

subcortical

on cyclic

and

astroglial

ACTH

increases

has

AMP cells

(8)

reported. is

reported

AMP

as

in well

as

$1.50 0 1985 by Acudemic Press, qf reproduction in an.y .form

of cyclic

Inc. reserved.

paper GMP in

286

that

cultured

?

protein

molecular

(7)

cl,21

of RNA and

elucidated.

neurons

system

phosphoryla-

shown

regulate

(ACTH)

involved

regulation

underlying

messenger

evidence

nervous

demonstrated

and

the

hormone

(3).

ACTH

remain

second

central

in

recently

neurons.

these

the

and

We

all

as

(61 and

It

behaviour

of

of

the

cyclic

31.

cultures

adrenocorticotropic

within

proteins

in

maturation

action

0006-291X/85 Copyrighr All rights

metabolism

pure

ACTH

of

widely

(reviewed

and

that

occurs

synaptic

val

granted

of phosphatidylinositol,

synthesis

have

for

chick

embryo

the

levels cortical

Vol.

133,

No.

1, 1985

neurons,

the

BIOCHEMICAL

hormone

being

AND

BIOPHYSICAL

lO,OOO-fold

more

RESEARCH

potent

in

COMMUNICATIONS

increasing

cyclic

GMP level. MATERIALS

AND

METHODS

and chemicals. Peptides Synthetic ACTH(l-24) was generously provided by Drs R. Andreatta and K. Scheibli (Ciba-Geigy, Basle, Switzerland). ACTH(l-10)) ACTHIL-101, ACTH(4-111, ACTH(l-131, a -melanocyte stimulating hormone ( a-MSH) and isobutylmethylxanthine were purchased from Sigma (St Louis, MO, USA). [y.?2P]-guanosine triphosphate, cyclic AMP assay kits and cyclic GMP RIA kits were from the Radiochemical Centre, Amersham (Les Ulis, France). Culture media and sera were from Flow Laboratories ( AsniPres, France 1. Falcon plastic culture dishes were used. Cultures of neurons from embryonic chick cerebral hemispheCell culture. res were maintained as described (4,5). Cultures were initiated and for 3 days in Dulbecco’s Modified Eagle’s Medium (DMEM) supplegrown mented with 20% fetal calf serum, penicillin (25 U/ml) and streptomycin From the fourth day, cultures were grown in serum-free (25 pg/mll. DMEM containing glucose (33 mM) , transferrin (100 ug/ml) , insulin (5 putrescin (0.1 mM), progesterone (10 nM), selenium (30 nM) pg/ml), and antibiotics as described (5). Cultures were grown for 7 days in 60 mm plastic dishes. Media were renewed at alternate days. Measurement

of

ACTH-induced

changes

in

cyclic

nucleotides

levels.

Seven-

day-old neuronal cultures were preincubated for 20 min in the absence of peptide, followed by incubation with various concentrations of ACTHIl24) or its related peptides. Incubations were carried out in 2 ml of Hepes-buffered Krebs-Ringer solution (KRH: NaCl 125 mM, KC1 4.8 mM, MgSO, 1.3 mM, KH ,PO, 1.2 mM, glucose 1 g/l, Hepes 25 mM, pH 7.4) containing 1 mM isobutylmethylxanthine (IBMX). The reaction was terminated by aspiration of the media and addition of 350 1.11 of ice-cold 1 M perchloric acid. Cellular proteins were centrifuged at 5,000 g for 15 min. The supernatants were neutralized with 3 M K, CO 3 and cyclic nucleotide content was determined with the cyclic AMP assay kit and the cyclic GMP RIA kit from Amersham. Protein was determined on the resuspended pellet by the method of Lowry. Guanylate cyclase assay. Cultures were incubated for 5 min with various concentrations of ACTH(l-241 in complete serum-free DMEM. Cells were then homogenized in 250 u 1 of ice-cold 10 mM Tris-HCl buffer, pH 7.5, containing 0.5 mM EDTA and 10 mM B-mercaptoethanol. Guanylate cyclase activity was measured as previously reported (9). RESULTS

Incubation ce

of

the

of chick

increase

trations

(figure cyclic

min)

neurons

phosphodiesterase

a rapid

The

cortical

AMP

of the 1).

with

Maximal

response

incubations,

while

a

Accumulation

of

cyclic

cyclic

AMP

stimulations

was

in

and

were

slightly

only

decline

in

the

isobutylmethylxanthine

inhibitor

intracellular

ACTH(l-24)

the

produced

cyclic

GMP concen-

achieved

after

decreased cyclic

GMP

presen-

with

6-8

min.

longer

(30

was

more

response

rapid.

of

ACTH(l-24)

treatment

is

nucleotides represented 287

in in

neurons figure

at 2.

The

various

doses

concentrations

Vol.

133,

No.

1, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

I

0

10 20 Time of incubation (inin)

10 Time of incubation

30

30

20 (min)

Time-course of ACTH-induced increase of cyclic AMP (A) and (B) levels in cultured neurons. Seven-day-old cultures were incubated in Hepes-buffered KRH, pH 7.4, in the presence (0) or in the absence (m ) of ACTH(l-24) (10 FM in A and 10 nM in B). All incubations were in the presence of 1 mM IBMX. After the indicated times, intracellular cyclic AMP and cyclic GMP contents were determined with the Amersham kits. Each point is the mean ? S.D. of 6-10 determinations.

:*

of

MP

ACTH

and

( l-24)

most

cyclic

GMP

respectively.

concentrations was

of

much

cyclase and

activity

served A to

shown

efficiency cyclase

number

affect

(at

of

containing

compounds

the

AMP,

but

less

than

tides

were

not

able

to

the

ACTH-peptides

ACTH(l-24)

(EC,,:

ACTH(4-lo), (l-10), and

five-fold

response. AMP

level,

the

0.5

p M)

less Furthermore, were

unable

found

in

(4-11) efficient

than

PM).

twenty-fold

affect

By

and cyclic

288

1

to a -MSH,

GMP

that

production.

while

ob-

rise. ability

in

increasing

all

former

pep-

potency the

1 J.IM)

induce

the

that

the

in

nM)

shows

The

potent

0.02

their

effective

less

:

GMP

Table

contrast,

guanylate

for

for

(EC5,,:

ACTH(l-24)

ACTH(l-13)

same

decreased

a-MSH

5-10

(ECSo

receptors.

higher

GMP

on

cyclic

suggests

response

cyclic

tested

were

ACTH

a ACTH(l-13), so:

the

neurons.

This

AMP

were

to

for

(4-10)

for

the

AMP

stimulation

potency

were

activate

cyclic

(EC

The

cyclic at

ACTH(l-24)

essentially

sequence

fully

for

and

of

ACTH(l-24).

ACTH(4-11) (4-10)

effects

peptides levels

cyclic

than

as

ACTH-related

nucleotides

cyclic

PM)

3.

for

declined

0.5

were

activation

nM

half-maximal

figure

1 nM)

1

for

(EC,,,:

in

and response

value

dose-response

are

guanylate

AMP

PM GMP

The

cyclic

The

100

cyclic

peptide.

for

nM).

maximal

The

the

higher

0.03

(EC,,:

were

efficacious

of

sequence:

>ACTH(l-lo),

ACTH (ECso the affecting

fragments := cyclic

10

nM) GMP cyclic

Vol.

133,

No.

BIOCHEMICAL

1, 1985

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

6-

i

y’12

11 - I09

10 [ACT,

9

6

7

l-24].(~)

=F

Concentration-response curves for the increase in the levels of cyc K AMP (0) and cyclic GMP (0) induced by ACTH(l-24) in cultured neurons. Cultures were incubated in KRH with the indicated concentrations of ACTH( l-24) in the presence of 1 mM IBMX. The intracellular contents of cyclic nucleotides were measured after incubation for 7.5 min. Each point is the mean * S.D. of 6 different cultures.

Figure 3. Concentration-response curve for the activation of guanylate cyclase in cultured neurons. Cultures were incubated with the indicated concentrations of ACTH(l-24) in serum-free DMEM for 5 min at 37°C. Homogenates in 10 mM Tris-HCl buffer, pH 7.5, containing 0.5 mM EDTA and 10 mM B -mercaptoethanol were used for determination of guanylate cyclase activity (9). Each point is the mean +_ S.D. of duplicate determinations of 4-6 different cultures.

TABLE Structure-activity on

relationship cyclic

nucleotide

1

for

various

proruction

in

Percentage

Peptide cyclic

AMP

ACTH-related cultured

increase

peptides

neurons

in: cyclic

GMP

ACTH ( l-24)

95

102

ACTH(

58

20

l-10)

ACTH(4-10)

49

18

ACTH(L-11)

57

20

ACTH(l-13)

63

NS

80

NS

NS

NS

a-MSH ACTH(

18-39)

Cultured neurons were incubated in the presence of the different peptides listed (concentration range: 1 nM-10 y M). The intracellular cyclic AMP and cyclic GMP contents were measured after incubation for 7.5 min at 37°C in the presence of IBMX. Values are the mean of ACTH-induced increases of cyclic nucleotide levels from 3-6 independent experiments. NS: not significantly different from control values.

289

6

5

Vol.

133,

No. 1, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

DISCUSSION The cyclic

present

GMP

results

formation

by

cerebral

hemispheres.

for

ACTH

in

the

reguIation

this

is

the

first

report

the

effect

of

of

These

two

and

responses

of

observed

in

messenger

adrenal

There

13).

is

a

in

cyclic

response

in showing

AMP.

The

our

neuronal

reported

in

rat

brain

neurons

(7)

and

rat

(ECaO:

10

potent This

may

types

(8)

be or

for

the

cultures

subcortical cells

to use

was

(81,

cyclic

forskolin

in in

animal

the

a

the

second

two

for

cyclic GMP

the

similar

cyclic to

rat

was,

in

cyclic

cultured

response

already

as

for

almost In

GMP

(reviewed on

ACTH(l-24) AMP

differences

of

(6).

cyclic

AMP

(0.5pM)

found

involve-

were

potency

value

tissue

inducing

attributed to

EC,,

on

cyclic

elicited

higher

estimated

ACTH

comprehended

response much

astroglial

nM)

fully

activity.

the

(12)

of

transient

concentration

suggesting

role

is

the

and

activity

the

gland

difference

in

cyclase

this

rapid

mediates

cyclase

of

role

(6.7,8),

GMP

ACTH(l-24)

Effect

although

ACTH(l-24)

for

AMP

cortex,

nM

thereby

guanylate

ACTH

nucleotides, than

and

for

1

desensitization.

(10,ll)

tissues

guanylate

at

a

cyclic a

and chick

suggested

that

stimulated

AMP

embryonic

nerve

caused

concentrations,

receptor-mediated

formation

and achieved

higher

in

evidence

cyclic

from have

levels

ACTH(l-24)

were

at

neurons

AMP

provide

GMP

of

reports

cyclic

neurons.

stimulation

cultured

several

to

in

the

in

of

cyclic

decreased

ment

ACTH Whereas

ACTH

accumulation

demonstrate

that

cortical

however,

than

in

species

our

system.

(7,8),

estimation

of

the

tempting

to

argue

more

in

ACTH

cell

effect

(7).

On exist

may of

the two

receptors

AMP acting increase existence

basis sets may

production. with

of

these

of be

The ACTH

of

of

ACTH

types

to

second

leading

type to

is

populations

adenylate of

receptors

seems

cyclase

concentration. come

290

in

cyclase

receptors

guanylate

Ca++ of

it

receptor

coupled

intracellular two

data,

from

the

neurons.

to

there One

responsible

activation, Further

that

for

operate

by

cyclic inter-

probably arguments

structure-activity

set

via for

the stu-

Vol. 133,No.

1, 1985

dies.

ACTH(l-24)

cyclic

AMP

nal

they

are

cannot

of

rat

has

brain

and

protein

tide

response

tion

of

cyclase

is

cellular response

kinase is

is

shown

ACTH(I-13)

our

and

form,

induce functiona

a-MSH,

GMP

on

populations

system

the of

on

its

stimulated

cyclic

regulate

C activity known

GMP

both

N-termi-

cyclic

response. same

AMP Whether

neuron,

neurons

in

of elevation

protein

kinase in

the

polyphosphoinositlde

(14) in

to be

(17).

GMP

involved

to

membranes

a substrate

and

distinct

in

COMMUNICATIONS

or

the

if

culture

presently. been

cyclic

are

two

synaptic

cyclic

to

RESEARCH

peptide

amidated

unable

by

be concluded ACTH

Whereas

receptors

expressed

BIOPHYSICAL

potent

C-terminal

were

types

AND

most

GMP.

and they

two

the

cyclic

acetylated

these

lated,

was

and

productton,

in

BIOCHEMICAL

the

and

in

adrenal

accompanied

cortex in

Recent

demonstration

protein

kinase

C (18)

achieved

by

is

C activation. cellular

adrenal (16).

cortex

(15)

Phosphoinosi-

many

cell

types

by

that

rat

brain

guanylate

suggests

that

ACTH

How

functions

the

metabolism

via

the

eleva-

intra-

phosphoinositide

cyclic

GMP,

once

accumu-

regulated

by

ACTH

remains

to be elucidated.

ACKNOWLEDGEMENTS We thank Mrs Marie-Odile and Dr Anant N. Malviya work was supported in part Malmaison, France).

Revel for help for critically by a research

with guanylate cyclase assays reading the manuscript. This grant from Ciba-Geigy (Rueil-

REFERENCES 1.

;: 4. 5. 6. 7.

8. 9.

10.

Brownstein, M. J. (1980) Neural peptides and neuronal communication, pp. 93-99, Raven Press, New York. Krieger, D.T. (1983) Science 222, 975-985. J. (1982) Physiol. Rev. 62, 977-1059. de Wied, D. and Jolles, Daval, J.L., Louis, J.C., Gerard, M.J. and Vincendon, G. (1983) Neurosci. Lett . 36, 299-304. Daval, J.L., Anglard, P., Gerard, M-J., Vincendon, G. and Louis, J.C. (1985) J. Cell. Physiol. 124, 75-80. Wiegant, V.M., Dunn, A-J., Schotman, P. and Gispen, .W.H. 119791 Brain Res. 168, 565-584. Weiss, S., Sebben, M. and Bockaert, J. (1985) J. Neurochem. 45, 869-874. Van Calker, D., Loffler, F. and Hamprecht, B. (1983) J. Neurothem. 40, 418-427. Zwiller, J. , Basset, P. and Mandel, P. (1981) Biochim. Biophys. Acta 658, 64-75. Perchellet, J .P. and Sharma, R.K. (1979) Science 203, 1259-1260. 291

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AND

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RESEARCH

COMMUNICATIONS

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