Regulation of cyclic GMP levels by neurotensin in neuroblastoma clone N1E115

Vol.

129,

No.

May

31,

1985

1, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

117-125

REGULATION OF CYCLIC GMP LEVELS BY NEUROTENSIN IN NEUROBLASTOMA CLONE NlE115 Shimon Amar, Jean Mazella, Frederic Checler, Patrick Kitabgi and Jean-Pierre Vincent' Centre

Received

de Biochimie

April

11,

du CNRS, Universite de Nice, 06034 Nice Cedex, France

Part

Valrose,

1985

lz5 I-labeled [monoiodo-Tyr31neurotensin to Intact The binding of neuroblastoma NlE115 cells and the effect of neurotensin on the intracellular 37°C and under concentration of cyclic nucleotides were studied at conditions of pH and ionic strength. The radiolabeled physiological neurotensin analogue bound specifically to differentiated cells with a dissociation constant of 0.75 nM and a maximal binding capacity of 45 fmol/106 Incubation of neuroblastoma cells with neurotensin in the presence of cells. calcium ions resulted in a transient increase of 10 fold over basal level of the intracellular cyclic GMP concentration. Half-maximal stimulation was Under identical conditions the cyclic AMP obtained with 2 nM neurotensin. concentration only decreased by 20-30 X. These results suggest that cyclic GMP 0 1985 is a second messenger of neurotensin in neuroblastoma clone NlE115. Academic Press, Inc.

Neurotensin fies

(pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu)

a variety

central where

of

nervous it

is

hypothalamic according

system

associated slices

to

with

the

synaptosomal

triggers

neurons

neurotensin

of questions

peptide

central

affinity

should

process

of

be answered

should

before

Neurotensin synaptic also

of all

physiological

in

brain

(2).

of

the

mammals

Depolarisation

of

immunoreactivity affects

the

membranes

peptidases

In spite the

the

neurotensin-like

and

(8,9).

neuromodulator

in

fraction

(3).

(6,7)

or

present

Rat brain

(4,5).

neurotensin

1. To whom correspondence

is

a release

receptors

inactivate

a neurotransmitter

The

'+-dependent

of

as

(1).

Ca

a

activity

efficiently

criteria

satis-

that these role

electrical

contain are data,

high able

to

a number

of neurotensin

in

be addressed.

ABBREVIATIONS: 4-(2-Hydroxyethyl)-1-piperazine HEPES, EGTA, ethylenediaminetetraacetate ; ether)-N,N,N',N'-tetraacetic acid.

ethanesulfonic ethylene glycol

acid ; EDTA, bis @-aminoethyl

0006-291X/85 117

$1.50

Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

129,

the

No.

1, 1985

central

biochemical

BIOCHEMICAL

nervous

system

effects

of

the

AND

could

peptide

be

BIOPHYSICAL

fully

on its

RESEARCH

For

understood.

target

nerve

COMMUNICATIONS

cell

example,

the

remain

essentially

binding

sites

unknown. We recently present

demonstrated

in homogenates

excitable

cell

line

cellular

events

its

neuronal

receptors.

increases cells

the and

neurotensin

that

to its

the

This are

clone

provides

modulated

cyclic specific

cyclic

neurotensin

neuroblastoma

by

We show in the

intracellular

that

specific

of differentiated (IO).

the

that

a useful

the

present

paper

is

mediated

an electrically

model

association

GMP concentration

GMP response

NlE115,

that in by

are

of

to

investigate

neurotensin

to

neurotensin neuroblastoma the

association

NlE115 of

receptors.

MATERIALS AND METHODS Drugs

and peptides Z-Pro-Prolinal(N-benzyloxycarbonyl-prolyl-prolinal) was a generous gift from Dr. Wilk (Department of Pharmacology, Mount SinaI School of Medicine, N.Y.). l,lO-Phenanthroline. was purchased from Merck. Neurotensin, acetyl neurotensin(&13), neurotensin(Y-13) and neurotensin(l-12) were synthesized (11) and kindly provided by C. Granier and .I. Van Rietschoten (Facultb de Mhdecine Nord, Marseille, France). (monoiodo-Tyr3 heurotensin (100 or 2000 Ci/mmol) was prepared by purification of the iodination products of neurotensin on Sulfopropyl-Sephadex C-25 (12). Cell

culture Neuroblastoma NlE115 cells were propagated in 75 cm2 Falcon tissue culture flasks and cultured at 37°C in Dulbecco's modified Eagle's medium (H21) supplemented with 10 % fetal calf serum, in a humidified atmosphere of 5 % CO -95 % air. The culture medium also contained penicillin (50 units/ml) and streptomycin (50 rig/ml). Differentiated cells used in binding and cyclic GMP assays were obtained as follows. Cells cultured in the nondifferentiated state were detached before confluence by incubation for 3 min in a 10 mM phosphate buffer containing 140 mM NaCl, 5 mM KC1 and 0.02 % trypsin. After centrifugation, the cellular pellet was washed and resuspended in 10 ml of culture medium. An aliquot (100 ~1) of this suspension was used for enumeration of cells with a Coulter counter. The cell suspension was distributed into Falcon 24-wells plates (lo5 cells/well) and allowed to grow for 48 h under the standard conditions described above. Cells were then induced to differentiate for 48 to 72 h (10) in a H21 medium containing 0.5 % fetal calf serum and 1.5 % dimethyl sulfoxide (13). Differentiated cells were reincubated for 4 h in normal culture medium before use in biochemical assays. Binding

experiments Monolayers of neuroblastoma NlE115 cells (about 3.105 cells/well) were incubated at 37'C with various concentrations of (monoiodo-Tyr'J neurotensin (100 or 2000 Ci/mmol) in a total volume of 200 ul of incubation buffer (25 mM HEPES-Tris, pH 7.4, containing 140 mM NaCl, 5 mM KCl, 1.8 mM CaCl,, 3.6 mM MgClz, 0.2 % bovine serum albumin, 4.5 g/l glucose, 1 mM l,lO-phenanthroline and 0.1 nM Z-Pro-Prolinal). In competition experiments, the incubation medium also contained different concentrations of unlabeled neurotensin or its 118

Vol.

129,

No.

1, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

synthetic analogues. After 30 min of incubation, the medium was removed by suction and the cell layer was rapidly washed twice with 1 ml of incubation buffer. Cells were suspended in 1 ml of 0.1 M NaOH and the radioactivity bound to the cells was counted with an Intertechnique C G 4000 gamma counter at a counting efficiency of 80 X. Nonspecific binding was determined in parallel experiments in the presence of an excess (1 JJM) of unlabeled neurotensin and subtracted from total binding to obtain the specific binding. Measurement of neurotensin-induced changes in cyclic nucleotide levels Neuroblastoma cells in multiwell plates (about 3.105 cells/well) were preincubated for 5 min in the absence of peptide then incubated for different times with various concentrations of neurotensin or its synthetic analogues. These incubations were carried out at 37'C in 200 nl of the buffer used in binding experiments. The reaction was terminated by aspiration of the medium and the cell layer in each well was extracted with 0.8 ml of a 2/3-l/3 (v/v) ethanol-5 mM EDTA, Extracts mixture of pH 7.4. were centrifuged and supernatants were evaporated in a speed vacuum concentrator. Extracts were reconstituted with a 50 mM Tris-HCl buffer, pH 7.5, containing 4 mM EDTA and their cyclic nucleotide content was measured with the cyclic AMP assay kit and the cyclic GMP RIA kit from Amersham.

RESULTS AND DISCUSSION The purpose nucleotides

are

receptors

in

binding

at

and

37°C

homogenates of

that

(10).

inhibitor

extensively

the

present

with

intact

to

of

the

described

below

and 0.1

proline of

the

neurotensin were

NlE115

and carried

nM Z-Pro-Prolinal. 119

its out

not

analogues. in

found

the

that

for

30 min by high

medium

of

(14),

1 mM 0.1

of

endopeptidase

We NlE115

as measured

and

did

by

cells

towards

to

strength.

we

incubation

peptide

cyclic

was necessary

inhibitor,

of

assay

Since

study,

the

of

of

degraded

iodopeptide

concentrations

properties

it

results

types

pH and ionic

is

the

both

cyclic

specific

compare

cells, of

metallopeptidase

inhibitor

to

of

its

conditions.

intact

Addition

and biochemical

l,lO-phenanthroline

In

(> 95 %> protection these

order

conditions

chromatography. a

complete

experiments

of

in a 75 % degradation'of

a specific

checked

use

[monoiodo-Tyr31neurotensin

liquid

In

to

measurements,

neurotensin

37'C

levels

neurotensin

experimental

the

that

Z-Pro-Prolinal,

have

involve

and

cells.

identical

intracellular of

nucleotide

recently 20

binding

NlE115

under

l,lO-phenanthroline,

an almost

the

physiological

resulted

performance

by

and under

at

incubation 37'C

out

was to show that

cyclic

measurements

demonstrated

at

work

neuroblastoma

be carried

nucleotide

this

modulated

experiments

should

work

of

nM

produced

degradation.

modify

the

We binding

Therefore, presence

all of

1 mM

Vol.

129.

No.

Binding

1, 1985

BIOCHEMICAL

parameters

of

AND

neurotensin

BIOPHYSICAL

receptors

in

RESEARCH

intact

COMMUNICATIONS

neuroblastoma

NlE115

cells The radiolabeled NlE115

cells

that

first-order

at

experiments

excess as

the

(Fig.

Linearity

sites.

radiolabeled

1

2

plot

3

4

absence

(Fig.

(total

of

5

nM

receptor

binding)

-0

class complex

10

0.75

20

B , fmol

Binding

which

binding

is

binding

either

in

a large

is

defined saturable

that

of

noninteracting

formed

between

nM and the

30

40

the

maximal

50

/ 10 6 cds

of 1251-labeled[monoiodo-Tyr3)neurotensin to NlE115 cells. cells (3 x IO5 cells/well) were incubated with increasing of radiolabeled neurotensin. After incubation for 30 min at radioactivity bound to cells was determined as described in and Methods”. Nonspecific binding (0) was measured in the of 1 PM unlabeled neurotensin. Specific binding (0) is the between total binding (0) and nonspecific binding. Each point the mean of quadruplicate determinations from two different

120

the

of

is

1B) demonstrates

the

time

iodoneurotensin

binding

a single

pseudo

radioligand

equilibrium

cells,

nonspecific

with

and its

the

of radiolabeled

The specific

constant

analogue

F.

Figure 1. Differentiated concentrations 37°C the “Materials presence difference represents experiments.

Scatchard

dissociation

neurotensin

the

to

an incubation

of

differentiated

and

associates

The

0

in

total

of the

of

of after

results

the

(1 vM).

between

(monoiodo-Tyr3)neurotensin binding

or

neurotensin

difference 1A).

number

binding)

unlabeled

was attained

shows

to intact

according

Independently

shown).

concentrations

a fixed

(nonspecific of

1

associated

differentiated

equilibrium

increasing

with

3)neurotensin

previously not

Fig.

in which

presence

been

binding

37°C.

incubated

[monoiodo-Tyr

(results

used,

30 min

were

had

kinetics

concentration of

analogue

Vol.

129,

No.

binding

BIOCHEMICAL

1, 1985

capacity

which

of

corresponds

NlE115 to

with

NlE115

detected

in undifferentiated

The affinity to

those

of

various

to the the

45 fmol

neurotensin

native

neurotensin

(10)

COMMUNICATIONS

ligand

bound

sites

per

neurotensin

for

analogues

of unlabeled

half-displacement

2). of

constant

of

its

in

per

lo6

cell.

receptors

cells,

As already could

the

receptor

not

be

amounts

concentration

of

radiolabeled

unlabeled

was compared

experiments

and constant

The

bound

NlE115

competition

peptides

(Fig.

dissociation

of

RESEARCH

binding

neurotensin

[monoiodo-Tyr3)neurotensin induces

BIOPHYSICAL

cells.

concentrations

that

is

homogenates

of

three

cells

27,000

observed

AND

involving of cells

unlabeled

ligand

peptide

(IC50)

peptide-receptor

and

is

related

complex

(Kd)

by

expression

\

/

* Kd

where

(Lx] 5o is

and K* d

is

the

The value under

of

the

constant

dissociation 0.75

the

native

from

compete

cells

even

at

concentrations

the

importance

interaction

between have

or nonneural of

of

that

of

been

with

the

neurotensin

and

in neuroblastoma

Incubation increase

of

NlE115 of

2.

value The

its

of

to

of [ L*)so

that

the

analogues

on the

in

to

data

of

NlE115

underline

sequence

NlE115

intracellular

neurotensin

binding

These

receptors

not

Neurotensin(l-12)

8-13

receptor

neurotensin

native

for

C-terminal

nM

nM, a value

lower.

2).

0.1

The affinity

of

100 times

was

dissociation

was 0.88

1 pM (Fig.

its

other

NlE115

complex.

calculated

complex

as

and

with

half-displacement

ligand-receptor

(monoiodo-Tyr3)neurotensin

neurotensin

origin.

the

Fig.

was about

integrity

(15.16)

nucleotides

and

at

Imonoiodo-Tyr3)neurotensin.

as high

observed

ligand

labeled

identical

of neurotensin(9-13)

of

the

above)

was

to

transient

of

labeled

neurotensin-receptor

was unabl'e

Effect

constant

neurotensin(8-13) that

results

free

conditions

different

acetyl

of

nM (see

experimental of

whereas

concentration

Kd was

significantly of

the

for

the

cells.

Similar

neural

(6,7,10)

level

of

cyclic

cells

cells

with

30 nM neurotensin

10 fold

over

basal 121

level

of

produced the

a rapid

intracellular

and cGMP

Vol.

129,

No.

1, 1985

BIOCHEMICAL

AND

Iog

BIOPHYSICAL

[peptide,

RESEARCH

COMMUNICATIONS

M]

and Figure 2. Competition between 1251-labeled~monoiodo-Tyr31neurotensin unlabeled neurotensin analogues for binding to NlE115 cells. (monoiodo-Tyr3)neurotensin (0.1 nM) was incubated with NlE115 cells (3 x lOa cells/well) and the indicated concentrations of neurotensin ( a), acetyl neurotensin(8-13) ( n ). neurotensin(9-13) ( 0) and neurotensin(l-12) ( 0). The radioactivity bound to cells was determined after incubation for 30 min at 37°C. Results are expressed in percentage of the maximal specific binding. the mean of quadruplicate determinations from two Each point represents different experiments.

concentration

(Fig.

progressively.

Although

calcium

by EGTA in

intracellular

insensitive

no

were

the

after

longer unable

stimulation

20-30

by

medium.

cGMP concentration

of

s then

detectable

It

decreased

after

themselves

was observed

incubation

2 min

to

modify

when CaCl,

should

also

nondifferentiated

of cGMP

was omitted

be noted

NlE115

that

cells

was

to neurotensin.

Concentration-response analogues

on

cGMP

Neurotensin(l-12) neurotensin

levels

was

unable

analogues

concentration

as

analogues

measured

stimulation

of

neurotensin(8-13) 4 times

was maximal

was ions

no neurotensin-induced

and replaced the

The response

cGMP stimulation

incubation. levels,

3).

more

NlE115

cells

induce

any

cGMP increase.

IO-fold

increase

the

the

peptide

cGMP

level

(EC aa = 0.5 potent

in

and

nM)

40 times

the

same

neurotensin.

as the

for

to

produced

native

curves

effect

of are

However,

the

neurotensin presented

(ECSo)

different.

neurotensin(9-13)

less

potent 122

the

potencies that

and

in

respectively

Fig.

basal

of

induced

4.

cGMP

these

two

half-maximal Thus,

(EC a0 = 80 r&i) than

its

two other

The of

concentration were

and

acetyl were

neurotensin

Vol.

129,

No.

1, 1985

BIOCHEMICAL

0

AND

20

BIOPHYSICAL

40

60

lime

RESEARCH

100

COMMUNICATIONS

300

, s*c

Figure 3. Time-course of neurotensin-induced cGMF’ stimulation in neuroblastoma clone NlE115. Differentiated cells (3 x 10 ’ cells/well) were incubated in a Tris-Hepes buffer, pH 7.4, containing 1.8 mM CaC12 in the presence (0) or in the absence ( n ) of 30 nM neurotensin. After the indicated incubation time, the cGMF’ cell content was measured with the Amersham kit. In control experiments, no neurotensin-induced cGMP stimulation observed with Was undifferentiated cells (0) or after replacement of CaC12 by EGTA in the incubation buffer ( 0). Each point is the mean of sextuplicate determinations from three different experiments.

itself

(EC

neurotensin suggesting

50 = was that

2 nM).

Results

maximal neurotensin

at

in

30 nM

4 also

Fig. and

decreased are

receptors

show

regulated

that at

the

concentration

higher by

efficiency

a

desensitization

mechanism.

Figure 4. Concentration-response curves for the cGMF’ stimulation induced by neurotensin and its analogues in neuroblastoma clone NlE115. Cells (3 x 105cells/well) were incubated with the indicated concentrations of neurotensin ( a), acetyl neurotensin(8-13) ( n ), neurotensin(g-13) (0). or neurotensin(l-12) ( 0). The cGMP cell content was measured after incubation for 25 s at 37°C. Each point is the mean of quadruplicate determinations from two different experiments.

123

of

Vol.

129.

No.

1, 1985

BIOCHEMICAL

Neurotensin NlE115

cells.

concentration

RESEARCH

small

variations

of

CAMP levels

A maximal

20-30

% decrease

of

the

was

observed

after

No further

a 20-30

in

basal of

of

effect

this

neuroblastoma

intracellular

s incubation

characterization

COMMUNICATIONS

CAMP

NlEl15

cells

with

was attempted

in

work. Results

presented

cGMP concentration neurotensin are

BIOPHYSICAL

produced

30 nM neurotensin. this

AND

above in

binding

of

occupancy.

neurotensin

affinities

correlated

their

The ability

of

neurotensin-induced

clone

NlE115

The cGMP level

receptors

of

to

the

neuroblastoma

receptor

devoid

show that

is

is

increase

a direct

consequence

of undifferentiated

insensitive

to

neurotensin

and

three

of

potencies

for

increasing

its

cells

neurotensin.

of that

Moreover,

analogues the

of the

are

strongly

intracellular

cGMP

concentrations.

was recently Most

reported

of the

with

our

differed

data own

apart

two studies

The same authors

were

carried

data

with

studying

to out ours.

at

hamper

attempts

comparison

of

occurs

messenger determine neurotensin

conclusion, of

O"C,

whether

this

the

EC 5. values

(17)

cells it

different

these

exposure

of

the

NlE115

peptide

here

neuroblastoma mechanism

receptors. 124

occurs

which present

ability

of

binding

experiments their

binding used

the

present

cells

under

neurotensin cells

that

cGMP is

NlE115.

We are

in other

cells

for may

study, identical

degradation

to NlE115

suggest

clone

neurotensin

conditions

In

by blocking

presented

translation

in

in agreement

cGMP production

processes.

and effect

was made possible

in

temperature

two

were

compare

neurotensin-stimulated

binding

during

to

cells (17).

to 2 nM in the

However,

difficult

NlE115

communication

on the

(18).

in

for

as compared

in an abstract

makes

correlate

results

neurotensin

communication

NlE115

which

neurotensin

which

In

to

and

conditions

readily

bind

binding

physiological

preliminary

reported

the

to

in a short

: 13 nM in

also

cGMP formation

and Richelson

from

Furthermore,

neurotensin

stimulate

in this

results,

(3H)-neurotensin

to

by Gilbert

presented

in the

work.

neurotensin

at 37°C. a second

now trying that

to

possess

Vol. 129, No. 1, 1985

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

ACKNOWLEDGMENTS We wish was supported the Institut the Fondation

to thank G. Clenet for expert secretarial assistance. This work by the Centre National de la Recherche Scientifique (ATP1057), National de la Sante et de la Recherche Medicale (CRE 846021) and pour la Recherche Medicale.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Nemeroff, C.B., Luttinger, D., and Prange, Jr, A.J. (1982) Handbook of Psychopharmacology, vol. 16, pp 363-467, Plenum Press, New York. Uhl, G.R., and Snyder, S.H. (1976) Life Sci. 19, 1827-1832. Iversen, L.L., Iversen, S.D., Bloom, L., Douglas, C., Brown, M., and Vale, W. (1978) Nature 273, 161-163. Young, W.S. III, Uhl, G.R., and Kuhar, M.J. (1978) Brain Res. 150, 431-4:35. Miletic, V., and Randic, M. (1979) Brain Res. 169, 600-604. Kitabgi, P., Carraway, R., Van Rietschoten, J., Granier, C., Morgat, J.L., Menez, A., Leeman, S., and Freychet, P. (1977) Proc.Natl.Acad.Sci.USA. 74, 1846-1850. Mazella, J., Poustis, C., LabbC, C., Checler, F., Kitabgi, P., Granier, C., Van Rietschoten, J., and Vincent, J.P. (1983) J.Biol.Chem. 258, 3476-3481. Checler, F., Vincent, J.P., and Kitabgi, P. (1983) J.Neurochem. 41, 375-384. Checler, F., Emson, P.C., Vincent, J.P., and Kitabgi, P. (1984) J.Neurochem. 43, 1295-1301. Poustis, C., Mazella, J., Kitabgi, P., and Vincent, J.P. (1984) J.Neu-rochem. 42, 1094-1100. Granier, C., Van Rietschoten, J., Kitabgi, P., Poustis, C., and Frey&et, P. (1982) Eur.J.Biochem. 124, 117-124. Sadoul, J.L., Mazella, J., Amar, S., Kitabgi, P., and Vincent, J.P. (1984) Biochem.Biophys.Res.Commun. 120, 812-819. Kimhi, Y., Palfrey, C., Spector, I., Barak, Y., and Littauer, U.Z. (1976) Proc.:Natl.Acad.Sci.USA. 73, 462-466. Wilk, S., and Orlowski, M. (1983) J.Neurochem. 41, 67-75. Kitabgi, P., Poustis, C., Granier, C., Van Rietschoten, J., Rivier, J., Morgat, J.L., and Freychet, P. (1980) Mol.Pharmacol. 18, 11-19. Kitabgi, P., Kwan, C.Y., Fox, J.E.T., and Vincent, J.P. (1984) Peptides 5, 917-923. Gilbert, J.A.. and Richelson, E. (1984) Eur.J.Pharmacol. 99, 245-246. Gilbert, J.A., McKinney, M., and Richelson, E. (1984) Soc.Neurosci.Abstr. 10, 378.

125