Neuropeptidcs (1988) 12, 141.148 @ Lon#nan Group UK Ltd 19%

Characterization of [3t-l] CCK4 Binding Sites in Mouse and Rat Brain

CHRISTlANE DURIEUX, DIDIER PELAPRAT, BRUNO CHARPENTIER, JEAN-LOUIS MORGAT” and BERNARD P. ROQUES

Dhpartement de Chimie Organique, U 266 INSERM, UA 498 CNRS, Facultk de Pharmacie, 4 av. de I’Observatoire, 75006 Paris. *D&partement de Biochimie, CEN Saclay, 91190 Gif sur Yvette. (Reprint requests to B. P. R.)

Abstract-We have investigated the possible occurrence of distinct CCKs and CC& binding sites in the brain by comparing the binding characteristics of i3H] CC& to those of the CCKs analogue, [3H] Boc (Nle2*,31]CCK 27_33 (BDNL-CCK;I). f3Hl CC& and i3H] BNDL-CCK, were shown to interact with mouse brain membranes with very similar maximal binding capacities 31.7 f 2.1 fmol/mg prot /KD = 3.78 + 0.47 nM) and 38.9 + 2.2 fmol/mg prot (KD = 0.26 f 0.02nM) respectively. The apparent affinities of five CCK analogues for the sites labelled by both probes were almost identical. Autoradiographic studies revealed that the distribution of i3H] CC& binding sites in rat forebrain was the same as that of 13H] BDNL-CCK7, with high densities of receptors in the cortex, nucleus accumbens, olfactory bulb and the medial striatum, moderate densities in the amygdala, the hippocampus, several nuclei of the thalamus and hypothalamus. However in the interpenduncular nucleus where there was moderate binding of [3H]BDNL-CCK7, no [3H]CCK, labelling was observed. These studies demonstrated the occurrence of one class of high affinity binding sites for 13H] CC& in mouse and rat brain, with characteristics similar fo those already reported with CCK33, CCKB and pentagastrin probes. Nevertheless the presence of a small amount of very high affinity binding sites for [3H]CCK4 cannot be excluded.

Introduction Several distinct forms of CCK have been charac-

Cholecystokinin (CCK), a peptide originally iso- terized in brain and peripheral tissue. The pre-

lated from gut (1) has been found within neurons dominant form of CCK found in the brain is the

in various areas of the central nervous system octapeptide carboxyl-terminal CCKs (5) which is

(2-4). likely to play a neurotransmitter or neuromodu- lator role (6). Although less abundant, the un- sulfated octapeptide (CCKs NS) and the carboxy- terminal tetrapeptide (CC&) are also present in

Date received 29 March 1988 the central nervous system (7-8). Specific binding Date accepted 6 May 1988 sites for CCKs in brain and pancreas membranes

141

142 NEUROPEPTIDES

have been characterized using various radio- labelled probes, including [‘251]CCKa3 (9-13), [l”SI]CCKs (14, 15) [3H] BDNL-CCK, (16) [3H] CCKs (17) and [3H] pentagastrin (18). CCK recep- tors have been found to be discretely distributed in the brain, the highest concentrations occurring in the cerebral cortex, olfactory bulb, nucleus accum- bens and hippocampus (17-21). There is now substantial evidence that binding sites in the brain exhibit different characteristics to those in the pancreas (10, 22). The ligand selectivity of the peripheral receptor can be correlated with the ability of CCK fragments to release amylase. Thus, compared to CCKs, CCK4 is a weak inhibi- tor of [1251]CCK33 binding to pancreatic receptors and while CCKs is very potent in stimulating amylase release, CC& is virtually inactive (23).

that CCKs and CC& interact with different binding sites in the CNS although studies carried out with t3H] pentagastrin seemed to indicate a common receptor for CCKs and CC& (3 1, 32). However, since pharmacological experiments used the native fragment CCK4, a more appro- priate radioligand for binding studies is the tritiated analogue (33).

In the brain, no clear correlation has been found between binding studies and various pharmaco- logical responses. In competition experiments the C-terminal tetrapeptide exhibits an apparent affinity close to that of CCKs for central receptors (11,17,18) however in various pharmacological or biochemical experiments the tetrapeptide has been found to induce either the same effect as CCKs, or an opposite effect, or to be inactive. Thus CC& produced a small increase in the number of [3H] spiperone binding sites in striatal membranes whole CCKs reduced the number of sites and increased their affinity (24). Moreover, while CCKs had a predominantly inhibitory effect on spike discharges of the nucleus tractus solitarius neurons, CCK4 either exerted an excitatory effect or was inactive (25). Likewise CCKa and CC& have opposite effects on intracranial self-stimu- lation behaviour (26) and, when injected into the nucleus accumbens, CCKs decreased locomotion and exploratory responses whereas CCK4 en- hanced them (27). On the other hand, both CCKs and CCK4 appeared to be potent excitants of CAi hippocampal pyramidal neurons (28). Finally, peripheral injection of CCKs inhibited locomoter activity and rearing in mice while CCK4 was inactive (29). However, Hommer et al. (30) reported that only CCKs was able to induce excitation of mesencephalic DA neurons, but both CCKs and CC& potentiated the inhibitory effects of the DA agonist apomorphine. An hypothesis which could account for these findings would be

We report in this paper the biochemical charac- teristics of the binding sites labelled by [3H] CCK4 in mouse brain. The binding characteristics of t3H] CC& were compared to those obtained with the CCKs analogue, [3H] BBNL-CCKT: Boc-Tyr- [S0aH)-[3H]file-Gly-Trp-[3H].&le-Asp-Phe-NH~ (20, 34) under identical experimental conditions. Finally the distribution of [3H] CCK4 binding sites in the rat brain was investigated by autoradio- graphy and compared to that previously described for [3H] BDNL-CCK, (20).

Materials and Methods

Chemicals

All the CCK-related peptides were prepared in the laboratory by liquid phase synthesis (35-37) t3H] BDNL-CCK,: Boc-Tyr(S03H)-[43H]Nle-Gly- Trp-(43H]Nle-Asp-Phe-NH2 (lOOCi/mmol) was synthesized as described elsewhere (38). L-3-(3’, 4’, 5’-tribromophenyl)alanine was used in the synthesis of [3H]CCK4 precursor. The reductive tritiation was performed using PdO as catalyst to yield [3H]CCK, with a specific activity of 35Ci/ mmol (33). The tritiated ligands were of >98% purity which was achieved, when necessary, by HPLC (Waters apparatus) on a k Bondapak Cis reverse phase column and U.V. (210nm) detec- tion using as eluent acetonitrile/25 mM triethyl- ammonium phosphate buffer, pH 3 for [3H]CCK4 and pH 6.5 for [3H]BDNL-CCK7 (flow rate 1.5 ml/ min) .

Tissue preparation

Crude membrane fractions were prepared as pre- viously described (16). Briefly, Male Swiss Webster mice (body weight: 20g) were killed by decapitation. Brain, minus cerebellum and brain- stem, was homogenized in 50 mM Tris-HCl buffer, pH 7.4 (12ml/g wet tissue). The homogenate was incubated for 30 min at 35°C and centrifuged for 35

CHARACTERIZATION OF [‘H] CCb BINDING SITES IN MOUSE AND RAT BRAIN 143

min at 100000 x g. This procedure was repeated and the pellet finally resuspended in incubation buffer (50mM Tris-HCl, 5mM MgClz, 0.2mg/ml bacitracin, pH 7.4) and used immediately.

Binding assays

Receptor binding assays for [3H]CCK, and [3H] BDNL-CCK, were carried out in 50mM Tris-HCl buffer (pH 7.4), 5mM MgC12, 0.2mg/ml baci- tracin, in a final volume of lml (0.5-0.7mg pro- tein/ml) at 25°C for 60 min. The specific binding of 13H] CC& and 13H] BDNL-CCKr was defined as the difference between the radioactivity bound in the presence and the absence of 1 p.M CC& or CCKR respectively. Samples were filtered over Whatman GF/B glass fiber filters preincubated for 60 min in incubation buffer supplemented with bovine serum albumin (BSA) 1 mg/ml, and washed twice ([3H] BDNL-CCK, experiments) or three times (3H] CC& experiments) with 5ml of ice cold buffer. The radioactivity bound to the filters was measured by liquid scintillation spectrophoto- metry, using 5 ml of Beckman Ready Solv. Protein concentrations were determined by the method of Lowry et al (39). Kr values were calculated by using the Cheng-Prusoff equation (40).

Autoradiography of [3H] CC& binding sites on rat brain sections

Rat brain sections (20km) were processed as previously described (20). Sections were pre- incubated for 30 min in 50mM Tris-HCl pH 7.4 containing 0.2% bovine serum albumin. The sections were dried in cold air and incubated at room temperature for 60 min with 4nM [3H] CCK4 in Tris-HCl pH 7.4 containing 5mM MgClz and 0.5mgIml bacitracin. Non-specific binding was determined in the presence of 1 +M CCK4. The sections were rinsed (2 x 10 min) in 50mM Tris, 0.2% BSA at 4°C and quickly dipped (2 x 5s) in ice-cold water. For biochemical characterization, sections were then wiped-off and counted.

For autoradiography, sections were dried in cold air and tightly juxtaposed to tritium-sensitive Ultrofilm (LKB) and stored for 6 months at -20°C. Quantification was carried out by densito- metry, using tritiated standards (20). All autoradi- ographic sections were analysed according to the atlas of Paxinos and Watson (41).

Results

Binding characteristics of [‘H]CCKd and [“H] BDNL-CCK7 to mouse brain membranes

The extent of [3H]CC& degradation by mouse brain tissue under the conditions used in the binding assay was checked by HPLC. After 60 min at 25”C, 90% of the radioactivity coeluted with unlabelled CCK+

The non-specific binding observed with [3H] CCK4 in tissue homogenates was generally higher than that obtained with [3H]BDNL-CCK7. Mouse brain homogenates were chosen for the biochemi- cal characterization of the [3H]CCK4 binding sites, as there was a lower percentage of non specific binding (-50%) than with rat homogenates (-65%) at the concentration used in displacement experiments (half saturation). These differences between species have also been reported for other ligands (31,42).

Binding of 4nM [3H] CCK4 to mouse brain tissue reached a steady-state within 10 min at 25°C with an apparent pseudo first order rate constant k + 1 (app.) of 0.38 min-l. Saturation experiments performed under steady state conditions (60 min, 25°C) showed that, between 0.8nM and 30nM. [3H] CCK4 interacted apparently with a single class of sites (Fig 1) characterized by the following parameters (mean + SEM) : ko = 3.78 2 0.47nM, Bmax = 31.7 k 2.1 fmol/mg of prot (n = 7). This

‘; 30

E”

; 20

,E

m IO

(

0 15 30 fmol x mg-l

3

[ 1 25

3H CCK4, nM 50

Fig 1 Binding isotherm of the specific binding of 13H] CC& to mouse brain membranes. A scatchard plot of the data is shown in the insert. The data shown are from a single representative experiment with each point in triplicate.

144

fmol xmg-1

0 1 2

I 3 3H BDNL-CCK7 , nM

Fig 2 Saturation curve of the specific binding of [3H] BDNL- CCK7 to mouse brain membranes, with the corresponding scatchard plot in the insert. The data shown are from a single representative experiment with each point in triplicate.

binding capacity was close to that obtained from saturation experiments performed in parallel with [3H] BDNL-CCK, in the concentration range 0.05 nM to 1 nM (Fig 2): Bmax: 38.9 + 2.2 fmol/mg protein. Ko: 0.26 + 0.02nM (n = 6).

The apparent affinities of CCKs and related peptides for mouse brain [3H]CC& and t3H] BDNL-CCK7 binding sites are given in the Table.

NEUROPEPTIDES

For each analogue tested, similar K1 values were obtained using either ligand. Thus, the radio K1 CC&/K1 BDNL-CCK, was around 1 for all deri- vatives. With both ligands the rank order of potency was BDNL-CCK, > compound II > CCKs > CCKsNS > CCK4 > compound I.

Autoradiographic distribution of [‘H]CCK4 bind- ing sites on rat brain sections

A lower non specific binding was obtained with brain sections as compared to homogenates and in the rat, the specific binding represented routinely 6570% of the total. As we have already reported the autoradiographic distribution of [3H]BDNL- CCK, binding sites in the rat (20) the same species was chosen to investigate the distribution of [3H] CCK4 binding sites, allowing a direct comparison to be made between the two species. The experi- mental procedure developed for [3H] BDNL- CCK7 (20) was readily applicable to [3H] CCK4. Preliminary biochemical studies on rat brain sections showed that, at room temperature, equili- brium for 4nM [3H]CC& was achieved within 45 min, and that the number of binding sites re- mained constant for at least 2 hours. An incu- bation time of 60 min was therefore chosen. Saturation performed with striatum sections gave an affinity constant K,,: 2.7 f OSnM, and a maximum number of binding sites (B,ax) of 62 + 12 fmol/mg protein, close to that found using [3H]BDNL CCK7 (Bmax = 57 fmoVmg protein, KD = 1.76nM) (20).

Table Comparison of the Inhibitory Effects of CCK Analogues on the Binding of [3H] CC& (4nM) and [3H]

BDNL-CCK-, (0.2nM) in Crude Mouse Brain Homogenates

KI (nM) KI CCK,

rHJ CCK, [‘HJ BDNL-CCK, K, CCK4

CCKs 1.17 + 0.42 0.94 f 0.04 1.24 BDNL-CCK, 0.48 + 0.10 0.35 + 0.09 1.37

CCK, 9.20 + 2.41 6.60 + 1.03 1.39 CCKBNS 5.30 + 0.31 4.18 f 0.05 1.26 Compound I. 25.51 k 5.30 25.00 f 4.4 1.02 Compound II. 0.54 f 0.04 0.60 + 0.03 0.90

I. Boc-y-D-Glu-Tyr(SO3H)-N1e-D-Lys-Trp-N1e-Asp-Phe-NH~ (36). II. Boc-Tyr(SO,H)-gNle-mGly-Trp-(N-Me) Nle-Asp-Phe-NH1 (37). The Kr values are the mean + SEM of three independent determinations, each in triplicate. Each KI value was obtained from computer analysis of Hill plots with 9 concentrations of unlabelled ligand. gNle-mGly represents a retro-inverso amide bond.

CHARACTERIZATION OF [‘HI CCK., BINDING SITES IN MOUSE AND RAT BRAIN 145

Figure 3 shows autoradiographic distribution of [3H]CC& binding sites at two levels of the rat forebrain. High densities of sites were found in the anterior cingulate cortex, layer IV of the fronto- parietal somatosensory cortex, the piriform cor- tex, the nucleus accumbens, the olfactory tubercles, the endopyriform nucleus and the medial part of the striatum (Fig 3A) and in the posterior cingulate cortex, the ventromedial hypo- thalamic nucleus, and the medial nuclei of the amygdala (Fig 3B). Moderate densities were found in all other layers of the neocortex (Fig 3A) as well as in the paraventricular thalamic nucleus, the thalamic reticular nucleus and various nuclei of the amygdala (Fig 3B). Moderate to low levels were found in the lateral part of the striatum (Fig 3A) and the hippocampus (Fig 3B). In this latter region, labelling was concentrated in the dentate gyrus and the subiculum. Moreover, high densities were also observed in the olfactory bulb, as well as in the entorhinal and retrosplenial cortices where- as moderate densities were seen in the anterior olfactory nucleus, the habenula and the supraoptic nucleus (not shown).

The relative amounts of binding sites labelled by [3H]CCK4 in different brain areas are shown in Figure 4, and compared to those obtained with [3H]BDNL. As can be seen from this figure there was a good correlation between the distribution of the two binding sites: for instance a high density of

13H]CCK 4

Fig 3 Comparative distribution of [3H] CC& (A, B) and [3H] BDNL-CCK, (C, D) binding sites in rat brain sections. The slide mounted rat brain sections were incubated with 4nM [3H] CC&, and processed as described in the methods section. C and D are taken from P6laprat et al. (20).

z ‘a 200

E” \ - 0 E

‘I

5 LS 0

F - K PA IP

___. I__ 0 100 200

[3~]~~~~ sites ,fmol/mg prot

Fig 4 Relation betweeen [3H]CC& and [3H]BDNL-CCK7 receptor densities in the rat brain. AA = anterior part of the nucleus accumbens. CC = cingulate cortex; DG = dentate gyrus; IP = interpenduncular nucleus; LS = lateral part of striatum; MS = medial part of striatum; OT = olfactory tubercle; PA = posterior part of the nucleus accumbens; PT = paraventricular nucleus of the thalamus; VH = ventromedial hypothalamic nucleus. In order to calculate B,,, values (assuming Ko values for [3H]CCK.+ and [3H]BDNL-CCK7 identical in all regions) bound values should be multiplied by 1.59 for BDNL-CCK, and 1.65 for CCK+

[3H]CCK4 binding sites was observed in the anter- ior region of the nucleus accumbens whilst the posterior region only contained low levels of binding. A similar rostro-caudal gradient in this nucleus was observed using [3H]BDNL (20).

The only region for which a discrepancy was observed was the interpeduncular nucleus in which [3H]CC& binding sites were undetectable, although moderate densities of 13H]BDNL bind- ing were measured.

Discussion

Since the presence of CC& has been reported in brain (8), specific receptors for this fragment might exist, and could account thus for the differences in pharmacological responses induced by CC& and CCKg. CC& is a potent inhibitor of the binding of various radiolabelled probes to brain CCK recep- tors, and it has been found that its affinity seems to differ between rodent species (42). However, even

146

in the same species, differences exist in the litera- ture in the reported affinities of this fragment for central receptors. For example, in the mouse brain Clark et al. (31) found the same affinity for CCKs and CC& whilst Williams et al. (42) found a 20 fold difference between these two fragments. A radiolabelled probe corresponding to the native CC& was therefore designed to investigate the binding sites of this short peptide. [3H] CCK4 was shown to exhibit a saturable binding on mouse brain homogenates and rat brain sections with a Ko value in the nanomolar range. The binding characteristics of [3H] CC& resembled those of the CCKs-related ligand [3H] BDNL-CCKT. The binding capacities of both ligands were very similar and the apparent affinities of CCK analogues for the sites labelled by either tritiated probe were almost identical.

The distribution of [3H]CCK, binding sites in the rat brain was also determined by autoradi- ography. As illustrated in Figures 3 and 4, this distribution, as well as the relative amounts of sites between structures, were found to be similar to those previously found for [3H]BDNL-CCK7 (20). In particular, the same subregional localizations were observed in cortex, hippocampus and nucleus accumbens. This distribution is in accor- dance with those already described using various probes (17-20).

Several authors have recently reported the pres- ence of two CCK receptor types in the rat brain, the first is widely distributed and relatively non specific for CCK fragments (type B) and the second (type A) exhibits a higher degree’ of specificity for CCKs and is localized in the area postrema, the nucleus tractus solitarius, and the interpeduncular nucleus (43,44). In the course of this study on rat forebrain, we noticed that inter- peduncular nucleus was not labelled by [3H]CCK+ This is in accordance with the very low affinity of CCK4 for the A site observed by these authors.

Thus, using the tritiated CCK4 we have found one class of binding sites in mouse and rat brains with characteristics similar to the B site already described with various CCKs and CCK33 related probes. This is in agreement with the results obtained by Clark et al. and Gaudreau et al. (31, 32), with [3H] pentagastrin in place of the native molecule. Nevertheless, we found a difference

NEUROPEPTIDES

between the values of the equilibrium dissociation constant of [3H] CCK4 (Ko: 3.78nM) and of inhibition constant of CCK4 (Kr = 9.2nM. It is interesting to note that this was not observed by Clark et al. (31), using [3H]pentagastrin in the same species. Such differences have previously been obtained with probes exhibiting moderate affinity and a low specific binding (45) and could be an inherant property of CCK4 binding. Another possible explanation for this apparent discrepancy could be the presence of a small amount of very high affinity binding sites for CCK4 which, owing to the relatively weak specific activity of [3H] CCK4, would be very difficult to demonstrate directly in saturation experiments. This situation was encountered with the opioid ~~ binding sites, a subtype of p receptors endowed with a very high affinity (46). However, if the pharmacological results showing similar actions of CCKs and CCK4 (28,30) can easily be explained by their interaction with common binding sites, those showing oppo- site actions for these two peptides (24-27) still remain to be explained at the receptor level. In this respect, high affinity binding sites for CCK4 could account for these latter effects, and this hypothesis deserves further investigation.

Acknowledgements

We wish to thank A. Beaumont for stylistic revision and A. Bouju for typing the manuscript. C. Durieux is in receipt of a fellowship from Rhone-Poulenc-Sante.

References

1.

2.

3.

4.

5.

Jorpes, J. E. and Mutt, V. (1966) Cholecystokininand pancreozymin one single hormone? Acta Physiol. Stand. 66: 196-202. Innis, R. B., Correa, F. M. A., Uhl, G. R., Schneider, B. and Snyder, S. M. (1979) Cholecystokinin octapeptide-like immunoreactivity: histochemical localization in rat brain. Proc. Natl. Acad. Sci. USA 76: 521-525. Larsson, L. I. and Rehfeld, J. F. (1979) Localization and molecular heterogeneity of cholecystokinin in the central and peripheral nervous system. Brain Res. 165: 201-218. Vanderhaeghen, J. J., Lotstra, F., De Mey, J. and Gilles, C. (1980) Immunohistochemical localization of cholecysto- kinin and gastrin like peptides in the brain and the hypophysis of the rat. Proc. Natl. Acad. Sci. USA. 77: 1190-1194. Rehfeld, J. F. (1978) Immunochemical studies of chole- cystokinin. Distribution and molecular heterogeneity of

CHARACTERIZATION OF [‘HI CCK, BINDING SITES IN MOUSE AND RAT BRAIN 147

6.

7.

8.

9.

10.

11.

12.

13

14

15

16

17.

18.

19.

20.

cholecystokinin in the central nervous system and small P. (1987) Autoradiography of CCK receptors in the rat intestine of man and hog. J. Biol. Chem. 253: 4022-4030. brain using [3H] Boc [Nle 28,31] CCKZ7_33 and [“‘I] Bolton- Morley, 1. E. (1982) The ascent of cholecystokinin (CCK) Hunter CCKs. Functional significance of subregional dis- from gut to brain. Life Sci. 30: 479-493. tributions. Neurochem. Int. 10: 495-508. Rehfeld, J. F. and Goltermann, N. R. (1979) Immuno- chemical evidence of cholecystokinin tetrapeptide in hog brain. J. Neurochem, 32: 1339-1341. Frey, P. (1983) Cholecystokinin octapeptide (CCK,.33), non sulfated octapeptide and tetrapeptide (CCK30_33) in rat brain. Analysis by high pressure liquid chromatography (HPLC) and radioimmunoassay. Neurochem. Int. 5: 811- 81.5.

21. Died, M. M., Probst, A. and Palacios, J. M. (1987) On the distribution of cholecystokinin receptor binding sites in the human brain: an autoradiographic study. Synapse I: 169-183.

22. Sakamoto, C., Williams, J. A. and Goldfine, I. D. (1984) Brain CCK receptors are stucturally distinct from pancreas CCK receptors. Biochem. Biophys. Res. Comm. 124: 497-502.

Hays, S. E., Beinfeld, M. C., Jensen, R. T.. Goodwin, F. K. and Paul. S. M. (1980) Demonstration of a putative receptor site for cholecystokinin in rat brain. Neuropep- tides 1: 53-62.

23.

Innis. R. B. and Snyder, S. H. (1980) Distinct cholecysto- kinin receptors in brain and pancreas. Proc. Nat]. Acad. Sci. USA, 77: 6917-6921.

24

Saito. A., Sankaran, H., Goldfine, I. D. and Williams, J. A. (1980) Cholecystokinin receptors in the brain: charac- terization and distribution. Science 208: 1155-1156. Saito. A.. Goldfine, I. D. and Williams, J. A. (1981) Characterization of receptors for cholecystokinin and related peptides in mouse cerebral cortex. J. Neurochem, 37: 483-490.

Jensen, R. T., Lemp, G. F. and Gardner. J. D. (1982) Interaction of COOH-terminal fragments of cholecysto- kinin with receptors on dispersed acini from guinea-pig pancreas. J. Biol. Chem. 257: 5554-559. Agnati, L. F., Fuxe, K., Benfenati, F.. Celani, M. F.. Battistini, N., Mutt, V.. Cavicchioli, L. Galli, G. and Hokfelt, T. (1983) Differential modulation and 5- hydroxytryptamine receptors in the brain of the rat. Neurosci. Lett. 35: 179-183.

25 Morin. M. P., de Marchi, P., Champagnat. J.. Vander- haeghen, J. J., Rossier, J. and Denavit-Saubie. M. (1983) Inhibitory effect of cholecystokinin octapeptide on neu- rons in the nucleus tractus solitarius. Brain Res. 265: 333-338.

Jensen, R. T.. Lemp. G. F. and Gardner, J. D. (1980) Interaction of cholecystokinin with specific membrane receptors on pancreatic acinar cells. Proc. Natl. Acad. Sci. USA 77: 2079-2083.

26.

Wennogle. L. P., Steel, D. J. and Petrack, B. (1985) Characterization of central cholecystokinin receptors using a radioiodinated octapeptide probe. Life Sci. 36: 1485 1492.

27.

Praissman. M., Martinez. P. A., Saladino. C. F., Berko- witz, J. M., Steggles, A. W. and Finkelstein, J. A. (1983) Characterization of cholecystokinin binding sites in rat cerebral cortex using a “‘1-CCKa probe resistant to degradation. J. Neurochem. 40: 1406-1413. Pelaprat, D.. Zajac. J. M., Gacel, G., Durieux, C., Morgat. J. L., Sasaki. A. and Roques, B. P. (1985) [3H] Boc [NleZs3’] CCKz7_33. a new highly labeled ligand for CCK receptors: binding on brain and pancreas. Life Sci. 37:2490.

28.

29.

30.

Van Dijk, A., Richards, J. G., Trzeciak, A., Gillessen, D. and Mohler. H. (1984) Cholecystokinin receptors: bio- chemical demonstration and autoradiographical locali- zation in rat brain and pancreas using [3H] cholecystokinin 8 as radioligand. J. Neurosci. 4: 1021-1033. Gaudreau, P.. Quirion. R., St. Pierre, S. and Pert, C. B. (1983) Characterization and visualization of cholecysto- kinin receptors in rat brain using [3H] pentagastrin. Peptides 4: 755-762. Zarbin, M. A.. Innis, R. B., Wamsley, J. K., Snyder, S. H. and Kuhar. M. J. (1983) Autoradiographic localization of cholecystokinin receptors in rodent brain. J. Neurosci. 3: 877~906. Pelaprat. D. ., Broer, Y., Studler, J. M., Peschanski, M., Tassin, J. P., Glowinski, J., Rostene, W. and Roques, B.

De Witte. P., Heidbreder, C.. Roques, B. P. and Vander- haeghen. J. J. (1987) Opposite effects of cholecystokinin octapeptide (CCKa) and tetrapeptide (CC&) after injec- tion into the caudal part of the nucleus accumbens or into its rostra1 part and the cerebral ventricles. Neurochem. Int., 10: 473-479. Katsuura. G., Itoh, S. and Hsiao, S. (1985) Specificity of nucleus accumbens to activities related to cholecystokinins in rat. Peptides 6: 91-96. Dodd, J. and Kelly. J. S. (1981) The actions of CCK and related peptides on pyramidal neurons of the mammalian hippocampus. Brain Res. 205: 337-350. Moroji. T. and Hagino, Y. (1986) A behavioural pharma- cological study on CCKa related peptides in mice. Neuro- peptides 8: 273-286. Hommer, D. W., Stoner, G., Crawley, J. N.. Paul, S. M. and Skirboll, L. R. (1986) Cholecystokinin-dopamine coexistence : electrophysiological actions corresponding to cholecystokinin receptor subtype J. Neurosci. 6: 3039- 3043.

31. Clark. C. R.. Daum, P. and Hugues, J. (1986) A study of the cerebral cortex cholecystokinin receptor using two radiolabelled probes: Evidence for a common CCKs and CCKj cholecystokinin receptor binding site. 1. Neuro- them. 46: 1094-1101.

32.

33.

Gaudreau, P., St. Pierre, S.. Pert, C. B. and Quirion, R. (1985) Cholecystokinin receptors in mammalian brain: a comparative characterization and visualization. Ann. N.Y. Acad. Sci. 448: 198-219. Charpentier, B., Roy, J., Morgat, J. L. and Roques, B. P. (1988) Synthesis of tritium labelled CCK30_33 (Trp-Met- Asp-[3H]3Phe-NH,), J. Lab. Compd. Radiopharm. (sub- mitted).

148

34.

35.

36.

37.

38.

39.

Durieux, C.. Coppey, M., Zajac, J. M. and Roques, B. P. (1986) Occurrence of two cholecystokinin binding sites in guinea pig brain cortex. Biochem. Biophys. Res. Commun. 137: 1167-1173.

Ruiz-Gayo, M., DaugC, V., Menant, I., Btgue, D., Gate!, G. and Roques, B. P. (1985) Synthesis and bio- logical activity of Boc [Nle 28, Nle”] CCK27_33. a highly potent CCKs analogue. Peptides 6: 415-420.

Charpentier, B., Pelaprat, D., Durieux, C., Dor, A., Reibaud, M., Blanchard, J. C. and Roques, B. P. (1988) Cyclic cholecystokinin analogs highly selective towards central receptors. Proc. Natl. Acad. Sci. USA, in press.

Charpentier, B., Durieux, C., Pelaprat, D., Dor, A., Reibaud, M., Blanchard. J. C. and Roques, B. P. (1988) Enzyme-resistant CCK analogs with high affinities for central receptors. Peptides, in press.

Sasaki, N. A., Funakoshi, S., Potier, P., Morgat, J. L., Genet, R., Gacel, G., Charpentier, B. and Roques, B. P. (1985) Synthesis of tritium labelled cholecystokinin deri- vative: [3H] Boc [Nle28.31]CCK2,_s~. J. Lab. Compd. Radiopharm. 22: 1123-1131.

Lowry, 0. H., Rosebrough, N. J., Fan, A. L. and Randall, R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275.

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41.

42.

43.

44.

45.

46.

NEUROPEPTIDES

Cheng. Y. C. and Prusoff, W. H. (1973) Relationship between the inhibition constant (K,) and the concentration of inhibitor which causes 50 percent inhibition (I,,,) of an enzymatic reaction. Biochem. Pharmacol. 22: 3099-3108. Paxinos. G. and Watson. C. (1986) The rat brain in stereotaxic coordinates. Academic Press. Sydney. Williams. J. A.. Gryson, K. A. and McChesney, D. J. (1986) Brain CCK receptors: Species differences in regional distribution and selectivity. Peptide. 7: 293-296. Moran, T. H., Robinson, P. H.. Goldrich, M. S. and McHugh, P. R. (1986) Two brain cholecystokinin recep- tors: implications for behavioural actions. Brain Res. 362: 175-179. Hill, D. R., Campbell, N. J.. Shaw, T. M. and Woodruff, G. N. (1987) Autoradiographic localization and biochemi- cal characterization of peripheral type CCK receptors in rat CNS using highly selective non peptide CCK anta- gonists. J. Neurosci. 7: 2967-2976. Delay-Goyet, P., Zajac, J. M.. Rigaudy, P., Foucaud, B. and Roques, B. P. (1985) Comparative binding properties of linear and cyclic &selective ligands. FEBS Lett. 183: 439-443, Lutz, R. A., Cruciani, R. A., Munson, P. J. and Rodbard, D. (1985) Mu,, a very high affinity subtype of enkephalin

binding sites in rat brain. Life Sciences 36: 2233-2238.