Occurrence of two cholecystokinin binding sites in guinea-pig brain cortex

Vol.

137,

June

30,

BIOCHEMICAL

No. 3. 1986

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages 1167-1173

1986

OCCURRENCE OF TN0 CHOLEXYSTOKSNIN BINDING SITES

IN GUINEA-PIG BRAIN CORTEX

Christiahe

DUBIEUX, Haitd COPPEY, Jeawharie and Bernard P. ROQUESf

ZAJAC

Dhpartement de Chimie Organique, UA 498 CNRS, II 266 INSERM, U.E.R. des Sciences Pharmaceutiques et Biologiques, 4 avenue de l’observatoire, 75006 PARIS, France Received

May 16,

1986

Saturation experiments of the highly potent cholecystokinin aMlOgUe ([%IBNDL-~c~, 100 Ci/mol) with guinea pig C3B1Boc(diNle28 3,)CCK2T+3 brain cortex ih a large concentration range (0.05 nh to 30 nh) show the presence of two different binding sites (A site : KD = 0.13 hh, Bm - 35 - 92 Pmol/ng). Both sites exhibit Pmol/mg ; B site : KD - 6.4 nH, Bdifferent sensitivity to sodium ions and therefore can be selectively investigated at [%llBDNJrtX~ concentration lower than 1 nM for the A site in Tris buffer and in Krebs buffer for the B site. The selectivity factors pB/pA of various CCK related peptides vary Prom 58 for CCK4 to 26 for CCK8 and 4 for the antagonist (Nle ) CCK~~2z;i$co~; occurrence of two different CCK binding sites 2813,' the explain biphasic pharmacological effects of CCK8. 0 1986 Academic Press, Inc.

The

sulfated

constitutes (1,2).

the Most

effects,

major

of the

with

Nevertheless, and

All

a single

characterized

and

these class

hyperlocomotion

observed and the

more

relationship

in

the

brain through

mammalian

have

affinity has

of

binding

experiments

(9-11).

potentiation

by CCKa of

apomorphine-generated

For

stereotypy

(4,5)

(7)

that

these

in brain

between

binding

example, the (12).

see

performed

CCK33 C3H]CCKg

sites

found

review

studies

concluded

been

by stimulation (to

binding

by use

brains

antistereotypic

structures

modif ied

recently

experiments of high

in

) seem to be mediated

in well-defined

pharmacological was

etc...

present

of CCKa (sedation,

Bol ton-Hunter

no clear

various

response

were

a (6)

(a).

pentagastrin interact

located

c125I1

C ‘2511imfdoester-CCK

fragment actions

potency,

receptors with

(Asp-Tyr(S03H)-Met-Gly-Trp-Met-Asp-PheNH2)

pharmacological

receptors

31. These

CCKg

cholecystokinin

anticonvulsant

of specific

mainly

peptide

dopamine

or

or

13H]

probes tissues.

studies a biphasic induced

Vol. 137, No. 3, 1986

These

results

have

been

detected

used

in

ligands

Using C3H]Boc(N1e this

the first

in

previous

specific

AND BIOPHYSICAL

an heterogeneity

experiments,

a new

studies of

of

with time

guinea the

CCK receptors

either

which

may not

concentration

radio-iodinated

probes

to

these

range,

or

tritiated

radioactivity.

tritiated

saturation

RESEARCH COMMUNICATIONS

due to the short

probe,

designed

28 ‘N1e31 )CCK27-33 (C3~I~~NL-CC~7,

study,

conditions

suggest

binding of low

BIOCHEMICAL

experiments pig

occurrence

cortical

loo Ci/mmol),

performed membranes

of two distinct

overcome

in which

(13)

thermodynamic demonstrate

CCK binding

sites

difficulties, we

report

in

equilibrium clearly

for

in brain.

MATERIALSANDMETRODS Chemicals [‘HJBDNL-CCK : Boc-Tyr(S0 H1-C3Hl Nle-Gly-Trp-C3H] Nle-Asp-Phe-NH (100 Ci/mmol) was synlhesized as des%ribed elsewhere (13). It4was of a puritc > 95 8, which was achieved when necessary by HPLC on p Bondapack C,8 column. The CCK-related peptides were synthesized in our laboratory. The gift from Dr J. Martinez antagonist (Nle 28 31 lCCK27-32 -NH2 was a generous (CNRS-INSERM Montp)ellier).

Binding experiments Crude membrane fractions of guinea pig brain cortex were prepared as Binding studies were carried out at 25V with previously described (14). freshly prepared membranes either in “Tris buffer” : 50 mM Tris HCI, 5 mM 0.2 mg/ml bacitracin (pH 7.4) or in “Krebs-Ringer buffer” : 118.5 mM W12, NaCl, 4.7 mM KCl, 1.2 mM KH2P04, 1.2 mM MgS04, 16.1 mM Na2HP04, 0.2 mg/ml Unless otherwise stated each assay (in triplicate) bacitracin (pH 7.4). contained 0.6 L 0.7 mg of protein in a final volume of 1 ml, the tritiated ligand at the indicated concentrations with or without 1 ~JM CCK8, in the presence or the absence of various competitors. Incubation (60 min. at 25OC) was terminated by rapid filtration through Whatman GF/B filters preincabated I1 buffers supplemented with bovine serum (60 min.) in “Tris” or “Krebs-Ringer Filters were rapidly rinsed with 2 x 5 ml of the cold albumin (BSA) 1 mg/ml. buffers, dried and the radioactivity was counted in 5 ml of Ready-Solv. EP scintillation cocktail (Beckman), with an efficiency pf 40 $. binding of Preliminary experiments have shown that steady-state [3~1~~~~-~~~7 (0.1 nM) was reached within 40 min incubation at 25’X in both buffers and remained at a plateau for up to 150 min. Saturation experiments were performed with ligand concentrations varying from 0.05 to 30 nM (12-16 concentrations). Isotopic dilutions with unlabelled BDNL-CCK7 were used in a concentration range from 6 to 30 nM.

Data analysis Data from saturation experiments were analyzed from Scatchard plots by or non linear regression analysis. In the latter case the program on the Gauss-Newton method, was adapted for a HP 85 computer. The fits were obtained by a series of iterations in which the parameters of the

linear based

1168

BIOCHEMICAL

Vol. 137, No. 3, 1986

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

saturation function for 2 or 3 independent sites were adjusted until a Displacement curves were fitted by least-squares solution was reached (15). the Hill transformation and KI values regression analysis of linear calculated according to Cheng-Prusoff equation.

RESULTS

Characterization brain

cortex.

membranes buffer

in was

The the

of two binding sites specific

binding

concentration

saturable

(fig.

of

range 1A).

BDNL-CCICI

in Guinea-pig

c3H1BDNL-CCK7 to guinea-pig -11 -8 t-l to 3 x 10 of 5 x 10

The

Scatchard

2

Bound, Figure

of [%I

r

fmol

representation

cortical M in Tris of

the

c

I mg-’

1.

Saturation analysis of [lHIBIRIL-CCK,blndlng to guinea-pig cortical membranes. A. Experimental points of a typical binding isotherm and theoretical binding curves assuming a two-sites model ( -) or a one-site model (- --) . B. Non linear regression analysis (two independent sites model) of the Scatchard plots of experimental data for [‘HIBDNL-CCK, binding in Tris-HCl buffer. KDA = 0.13 nM, KDB = 6.4 nM. C. Scatchard plots of [‘HIBDNL-CCK, binding : (D ) in Tris buffer (0.05 - 1 nm), K D - 0.17 nM ; (A) in Krebs buffer (0.5-30 nM), KD = 6.8 nM.

1169

Vol. 137, No. 3, 1986

BIOCHEMICAL

experimental

points

heterogeneity

of

independent sites function

was curvilinear

protein

suggesting

C3~l~~~~-C~~7 binding model, non-linear

(average of five

following

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

sites

the

(Fig.

regression

Occurrence

1B).

analysis

an

Assuming a two

of the saturation

independent experiments in triplicate)

parameters : KD - 0.13 + 0.09 nM , Bmax = 35.4 + (A site)

of

yielded the 8.1 fmol/mg of

and KD = 6.4 z 3.8 nM, Bmax = 92.6 + 20.3 fmol/mg (B site).

The binding parameters obtained from the non-linear the same experimental

regression analysis of

data assuming a one-site model were : KD = 1.62 + 0.89

nkf, Bmax = 106.4 2 9.2 fmol/mg. All independent experiments performed in this buffer

were significantly

172, p <

better

fitted

by a two independent sites model (F >

than a one site model. This is clearly

0.01)

illustrated

by the good

agreement between the average experimental points and the theoretical curves corresponding to the parameters calculated (fig.

binding sites. the B site labelled

conditions

for

exhibited

Scatchard

40.1

2

of

the

binding,

high affinity

lower than 1 nM. Thus, experiments

disappeared without

Krebs-Ringer,

(Fig.

(KD

component of

1C)

= 0.17

+

saturation

corresponding to the high affinity

50 % of high affinity in

could be

range of the tritiated

In Tris buffer,

Furthermore,

the A site

were close to those, computed from the

70 $ at the KD concentration. sites

the A site than for

range 5 x 10 -’ ‘M to 1 x lo-‘M

fmol/mgf,

1 .2

representation,

1B). The specific

buffer,

from saturation

experiments performed with a wide concentration (Fig.

for

at ligand concentrations

parameters calculated

nM, Bmax =

higher affinity

by C3HlBDNL-CCK7in Tris

almost selectively

binding

the Independent study of both C3H1BDNL-CCK’r

Owing to the 50 fold

performed in the concentration 0.03

assuming a two sites model

1A hatched line). Experimental

the

binding

non-linear

ligand site,

was

in the presence of 120 mMNaCl, any change in the B sites.

analysis

of

[3~1~~~~ binding

experiments (0.5 to 30 nM) revealed the presence of one single class of site exhibiting (fig.

lC),

a KD of 6.8 +

1.5

nM and a Bmax value of 118.9 + 15.5 fmol/mg

parameters which are closely

evidenced in Tris buffer

(Fig.

lB),

related

to those of

the B site

suggesting that the low affinity 1170

site of

Vol.

No. 3, 1986

BIOCHEMICAL

TABLE 1. Inhibition

of C31iIBDwL-CC~

137,

cortical

AND

BIOPHYSICAL

COMMUNICATIONS

to A and B sites

binding

membranes by CCK related A site

RESEARCH

of

guinea-pig

compounds

a)

B site

b,

KIB/KIA

Inhibitor KI (nM)

KI (nM)

Boc-UiNle28,3,-CCK27-33

0.13

+ 0.03

6.4

z 3.8

CCK26-33 (CCK8)

0.34

+ 0.07

9.0

+ 3.4

unsulfated

6.05

z 0.90

CCK26-33

CCK30-33 (CCK4)

+

22.60

diNle28,3,-CCK27-32-NH2

560.00

97.0 +

')

53

26

20

16 58

12

1300.0

5

400

+_ 60

2400.0

+

280

4

Results are averaged values (: S.E.M.) of three independent determinations in triplicate. a) in Tris-HCl buffer C3H]BDNL-CCK7 : 0.1 nM. b, in Krebs buffer experiments. C3H]BDNL-CCK7 : 1 nM. ‘) From saturation

C3H]BDNL-CCK7

was insensitive

high

hydrophobicity

site

was only

of BDNL-CCKT,

25 $ at half

Pharmacological apparent been

of

selectively

buffer the

KI

identical

to

its

of

saturation

conditions.

binding

of

the

two binding peptides

with B site

(A site

not

detectable)

the

A site

for

<

equilibrium

dissociation

experiments,

Due to the

of

A site

different

is

about

binding

different

peptides

apparent 26

affinity

times

sites

of

1).

of

higher

the

two

the for

of

binding

the

in

B

7

the

=

(KD = 0.13

of non specific

than

Indeed,

properties

native

for

sulfated

the B site.

appears

although 1171

the

by studied

have buffer nM in

1

0.13

1).

As

nM)

was

nM) computed interactions range,

by

CCK6

The relevance

several peptides

the

KI

displacement

peptide

to be strongly

elicited

Tris

(Table

(KI

The

sites

and with

B site

BDNL-CCK

presence

site

A

constant

[3H]BDNL-CCK7

discriminating (Table

to the

C3~l~~~~~~7.

C3H]BDNL-CCK7

8). for

10

sites for

nM

0.1

the

value

The

Due to the

corresponding

with the membrane preparation in the micromolar 7 BDNL-CCK 7 for the B site, cannot be determined

experiments.

the

ionic

BDNL-CCK

value

the

the

the specific

CCK related

with

expected

from

of

determined

(cross-reactivity

in

saturation.

profile

affinities

Krebs-Ringer

to changes

for

of two

supported CCK related interacted

by

Vol. 137, No. 3, 1986

preferentially varied

with

CCK,, to 4 for

were close to unity

binding to both sites were

0.6

site,

RESEARCH COMMUNtCATIONS

the selectivity

KIB/KIA

(diNle

the antagonist

for CCKaand related

for

peptides suggesting homogenous

these compounds. Contrastingly

found

for

CCK4 suggesting

Hill

to CCK such as opioid peptides,

coefficients

an heterogeneity

displacement of L3HI BDNL-CCK7from both A and B sites. unrelated

factors

28, 31 ) CCK27-32-NH2’ derived from displacement of C3~1~~~~-~~~7from A or B

coefficients

around

AND BtOPHYSICAL

the high affinity

from 58 for

The Hill sites

BIOCHEMICAL

in

Finally

the

compounds

neurotenain and SP were unable to

displace the c3H] BDNL-CCK7from A and B sites.

DISCUSSION Although

an heterogeneity

from biphasic results

dissociation

are the first

equilibrium,

of central

kinetics

direct

of various

has been suggested

probes

in guinearpig

cortex

of two different

This was made possible due to the high specific

of C3H] BDNL-CCK7which allowed the binding properties be investigated

over wide concentration

of two different

CCK binding sites

the present

(7,16),

in condf tions of thermodynamic

demonstration,

of the occurrence

binding sites.

CCK receptors

range (9).

CCK

radioactivity

of this new agonist to

The biological

remains to be firmly

relevance

established since in

the presence of a physiological

concentration

affinity

disappears. On the other hand, due to the

binding site selectively

inaccuracy

in

the .determination

of

the

of NaCl

concentration

occurrence of a sodium dependent interconversion sites

cannot be excluded.

distribution

of

situation.

both

Nevertheless,

CCK related

binding

of

stereotypies

after

contralateral

turnings

(17).

a precise

in

brain,

should clarify

the

discriminative

Moreover

administration after

in

direct

these features

properties

potentiation the rat

injection

seem to

1172

regional this

of various

suggest the existence of

which could account for

CCKa, such as

the

mechanismbetween A and B

peptidea towards A and B sites strongly

effects

B sites,

including

sites

sites

of

mM) the high

studies,

the different

two independent CCK binding biphasic

Further

(120

of

the well-known

dopamine induced

nucleus accumbens (12)

or

in the caudate nucleus of mice

be supported by a dose-dependent

Vol.

137,

BIOCHEMICAL

No. 3, 1986

modulation

of

dopamine

present

results

reported

dissociation

probably

due

binding

suggest

to

their

that

constants different

AND

sites the (0.2

BIOPHYSICAL

induced relatively

RESEARCH

by CCK8 large

to 2 nM) of various

degrees

of selectivity

(18).

range

COMMUNICATIONS

Finally, of

previously

CCK radioligands for

the

are

each CCK binding

site. ACKNOULEDGMEUTS and G. Gacel for the synthesis of the We thank Drs. 8. Charpentier acknowledged for peptides used in this study. Dr. A. Beaumont is greatfully her help in the preparation of the manuscript and A. Bouju for typing it. This work uas supported by grants from Rhone Poulenc Sant6, the Fondation the Ligue Nationale Franc;aise contre le pour la Recherche Mkdicale Franpaise, Cancer. C. Durieux is in receipt of a fellowship from Rhone Poulenc Sante. REFERENCES 1.

2. 3. 4. 5. 6. 7. 8. 9.

10.

11. 12.

13.

Vanderhaeghen, J.J., Lotstra, F., De Mey, J. and Gilles, C. (1980) Proc. Natl. Acad. Sci. USA ‘Q, 1190-1194. Beinfeld, M.C. (1983) Neuropeptides 3, 411-427. Gaudreau, P., St Pierre, S., Pert, C.P. and Quirion, R. (1985) Ann. N.Y. Acad. Sci. 448, 198-219. Innis, R.B. and Snyder, S.H. (1980) Proc. Natl. Acad. Sci. USA II, 6917-6921. Saito, A., Goldfine, I.D. and Williams, J.A. (1981) 3. Neurochem. 37, 483-490. Praissman, M. Martinez, P.A., Saladino, C.F., Berkowitz, J.M., Steggles, A.W. and Finkelstein, J.A. (1983) J. Neurochem. 2, 1406-1413. Van Dijk, A., Richards, J.G., Trzeciak, A., Gillessen, D. and Mohler, H. (1984) J. Neurosci. 4, 1021-1033. Gaudreau, P., QuirTon, R., Saint-Pierre, S. and Pert, C.B. (1983) Peptides 4, 755-762. Roques, B.P., Durieux, C., Gacel, G., Pelaprat, D., Ruiz-Gayo, M., Belleney, J., Fellion,E., Zajac, J.-M., Fournie-Zaluski, M.C., Daug6, V ., Menant, I., Rossfgnol, P., Lux, B., Gerard, D., Beg&, D., Sasaki, A. and Morgat, J.L. (1985) Ann. N.Y. Aca. Sci. 448, 61-75. Crawley, J.N. (1985) Ann. N.Y. Acad. Sci. 438, 283-292. Van Ree, J.M., Gaffori, 0. and De Wied, D. (1983) Europ. J. Pharmacol. 93, 63-78. Crawley, J.N., Hommer, D.W. and Skirboll, L.R. (1984) Neurochem. Int. 6, 755-760. Sasaki, N.A., Funakoshi, S., Potier, P., Morgat, J.L., Genet, R., Gacel, G Charpentier, B. and Roques, B.P., J. Labelled Compd. Radiopharm, 2, 1;;3-1133.

14.

Pelaprat, D., Zajac, J.-M., Gacel, G., Durieux, C., Morgat, J.L., Sasaki, A. and Roques, B.P. (1985) Life Sci. 2, 2483-2490. P., Roques, B.P. and Zajac, J.-M., Brit. J. Pharmacol., in 15. Delay-Coyet, press. 16. Wennogle, L.P., Steel, D.J. and Petrack, B. (1985) Life Sci. 3, 1485-l

492.

J., Briet, C., Castro, B. and Biziere, K. (1986) 17. Worms, P., Martinez, Eur. J. Pharmacol. 121, 395-401. 18. Fuxe, K., Agnati, L.F., Benfenati, F., Cimmino, M., Algeri, S., Hokfelt, T. and Mutt, V. (1981) Acta Physiol. Stand. 113, 567-569.

1173