Proc. Nadl. Acad. Sci. USA Vol. 90, pp. 65-69, January 1993 Pharmacology Biochemical and pharmacological profile of a potent and selective nonpeptide antagonist of the neurotensin receptor DANIELLE GULLY*t, MARYSE CANTON*, ROBERT BOIGEGRAIN*, FRANCIS JEANJEANt, JEAN-CHARLES MOLIMARD*, MARTINE PONCELETt, CHRISTIANE GUEUDET*, MICHEL HEAULMEf, ROGER LEYRIS*, ALINE BROUARD§, DIDIER PELAPRAT§, CATHERINE LABBt-JULLIO¶, JEAN MAZELLAI, PHILIPPE SOUBRIE*, JEAN-PIERRE MAFFRAND*, WILLIAM ROSTENE§, PATRICK KITABGI¶, AND GERARD LE FUR* Sanofi Recherche, *195 route d'Espagne, 31036 Toulouse C6dex, and t371 rue du Pr. J. Blayac, 34082 Montpellier C6dex, France; §UnitM 339, Institut National de la Sante et de la Recherche Medicale, H6pital St. Antoine, 184 rue du Fbg St. Antoine, 75571 Paris C6dex 12, France; and lInstitut de Pharmacologie Moleculaire et Cellulaire du Centre National de la Recherche Scientifique, Universit6 de Nice-Sophia Antipolis, Sophia Antipolis, 660 route des Lucioles, 06560 Valbonne, France Communicated by Susan E. Leeman, August 6, 1992

ABSTRACT We describe the characteristics of SR 48692, lines of peripheral and central origins (for a review, see ref. a selective, nonpeptide antagonist of the neurotensin receptor. 14). So far, structure-activity studies have not provided In vitro, this compound competitively inhibits 125I-labeled conclusive evidence for the existence ofneurotensin receptor neurotensin binding to the high-affinity binding site present in subtypes (14). However, adult rat and mouse brains have brain tissue from various species with IC50 values of0.99 ± 0.14 been shown to contain, in addition to high-affinity neuroten- nM (guinea pig), 4.0 ± 0.4 nM (rat mesencephalic cells), 7.6 ± sin receptors, low-affinity levocabastine-sensitive binding 0.6 nM (COS-7 cells transfected with the cloned high-affinity sites (15, 16). Whether these sites could mediate some rat brain receptor), 13.7 + 0.3 nM (newborn mouse brain), neurotensin effects remains to be elucidated (16). The main 17.8 ± 0.9 nM (newborn human brain), 8.7 + 0.7 nM (adult transduction system coupled to high-affinity neurotensin human brain), and 30.3 ± 1.5 nM (HT-29 cells). It also receptors in a variety of systems appears to be the guanine displaces 12-I-labeled neurotensin from the low-affinity levo- nucleotide-binding regulatory protein-dependent stimulation cabastine-sensitive binding sites but at higher concentrations of phospholipase C leading to an increase in intracellular (34.8 + 8.3 nM for adult mouse brain and 82.0 ± 7.4 nM for calcium (17). Recent cloning, sequencing, and expression of adult rat brain). In guinea pig striatal slices, SR 48692 blocks the high-affinity levocabastine-insensitive neurotensin recep- K+-evoked release of [3H1dopamine stimulated by neurotensin tor from rat brain has indeed revealed that it belongs to the with a potency (IC50 = 0.46 - 0.02 nM) that correlates with its family of guanine nucleotide-binding regulatory protein- coupled receptors (18). binding affinity. In a cell line derived from a human colon Despite the synthesis of neurotensin analogues, none of carcinoma (HT-29), SR 48692 competitively antagonizes neu- these compounds exhibited antagonist properties (14). The rotensin-induced intracellular Ca2+ mobilization with a pA2 present report describes the biochemical and pharmacological (-log Kpp) values of 8.13 ± 0.03, which is consistent with properties of SR 48692 {2-[(1-(7-chloro-4quinolinyl)-5-(2,6- results obtained in binding studies. Moreover, SR 48692 is dimethoxyphenyl)pvrazol-3-yl)carbonylamino]tricyclo(3J.;. devoid of any intrinsic agonist activity. This compound is also 1.1.3-7)decan-2-carboxylic acid}, active in vivo, since it reverses at low dose (80 pg/kg) the turning behavior induced by intrastriatal injection of neuro- tensin in mice with similar potency whatever the route of administration (i.p. or orally) and with a long duration of action (6 hr). Thus, being a potent and selective neurotensin receptor antagonist, SR 48692 may be considered as a powerful tool for investigating the role of neurotensin in physiological and pathological processes. Neurotensin (

65 Downloaded by guest on September 27, 2021 66 Pharmacology: Gully et al. Proc. Nati. Acad. Sci. USA 90 (1993) Table 1. IC50 values and Hill coefficients (nHJ for the inhibition of specific 125I-neurotensin binding by unlabeled neurotensin and SR 48692 in various species Neurotensin SR 48692 Sample ICso, nM nH IC50, nM nH Adult guinea pig brain 2.8 ± 1.6 0.95 ± 0.05 0.99 ± 0.14 1.15 ± 0.13 Adult rat brain 3.2 ± 0.5 0.89 ± 0.03 82.0 ± 7.4 0.67 ± 0.09 Rat mesencephalic cells 0.63 ± 0.16 0.89 ± 0.06 4.0 ± 0.4 0.91 ± 0.07 Transfected COS-7 cells 0.15 ± 0.02 0.74 ± 0.10 7.6 ± 0.6 0.95 ± 0.14 Newborn mouse brain 0.30 ± 0.02 0.86 ± 0.08 13.7 ± 0.3 0.97 ± 0.09 Adult mouse brain 2.0 ± 0.0 0.92 ± 0.06 34.8 ± 8.3 0.51 ± 0.08 Newborn human brain 0.31 ± 0.04 0.81 ± 0.09 17.8 ± 0.9 1.05 ± 0.12 Adult human brain 1.6 ± 0.4 0.75 ± 0.09 8.7 ± 0.7 0.83 ± 0.06 HT-29 cells 0.26 ± 0.04 0.75 ± 0.13 30.3 ± 1.5 1.01 ± 0.16 Each value represents the mean ± SE from at least three separate experiments performed in triplicate. described (19). Neurotensin was purchased from Neosystem technique as described (19, 21). Binding experiments with rat Laboratories. All other-chemicals were from commercial mesencephalic neurons (500,000 cells per well) were carried sources. out at 37TC as reported (20). Cel Culture. Mesencephalic cells from brains of embry- Auftradkgrapic Studies. Coronal sections (20 Aum thick) onic Wistar rats (day 15) and the human colon carcinoma from guinea pig midbrain were incubated with 1251 HT-29 and the mammalian fibroblast COS-7 cell lines were neurotensin and processed for film autoradiography as re- grown as described (20, 21). ported for rat brain sections (24). Tran1eto of COS-7 Cells. The HindIII-Not I fragment S rso Experimnts. Striatal slices from male guinea coding for the high-affinity rat brain receptor (gift of S. pig (350 Am) were preincubated 30 min with 120 nM Nakanishi; see ref. 18) was ligated into the HindIII-Not I 13Hldopamine in Krebs buffer containing 1 uM pargyline, 1 cloning site ofthe CDM 8 vector and transfected into COS-7 mM ascorbic acid, and 0.1 AM desipranine, saturated with cells by the DEAE-dextran precipitation method (22). 5% C02 in 02, and then superfused in a chamber at a rate of Pre~ara~on of Timms. Whole brain homoge- 0.5 ml/min with Krebs buffer containing bacitracin (40 mg/ nates from 7-day-old and adult mice, guineapig, adult rat, and liter), 5 mM dithiothreitol, and 0.1 mM 1,10-phenanthroline. newborn human (whole brain ofa 32-day-old male infant who After a 45-min wash period, two 3-min fractions were col- died from sudden infant death syndrome and dissected out lected to measure basal release. Neurotensin was introduced less than 48 hr after death) and cortical regions of an adult the fifth period and was presentuntil the end male human brain (dissected 10 hr after death) were prepared during collection as reported (23). Cell homogenates from confluent HT-29 of the experiment. SR 48692 (0.1-10 nM) was added 6 mmn cells were prepared as described (21). Membranes from prior to neurotensin. Release of [31Hdopamine was stimu- COS-7 cells were prepared similarly 48-72 hr after transfec- lated by superfusion for 3 min with buffer containing 20 mM tion. K+. Binding Assays. All binding assays with tissue homoge- Ca2+ Measurements. Subconfluent plated nates were carried out at 209C in 50 mM Tris HCl buffer (pH HT-29 cells were loaded for 90 min at 370C with 5 AtM indo-1 7.5) containing 0.2% bovine serum albumin, and 1 mM AM in complete culture medium. The cells were trypsinized, 1,10-phenanthroline (newborn mouse and human brain, washed, and diluted in incubation medium (140 mM NaCI/5 COS-7 cells, and HT-29 cells) or 0.1% bovine serum albumin, mM KCl/0.9 mM MgCl2/1.8 mM CaCl2/5 mM glucose, pH bacitracin (40 mg/liter), 1 mM EDTA, 5 mM dithiothreitol, 7.4) to about 50,000 cells per ml. Indo-1 fluorescence was and 1 mM 1,10-phenanthroline (guinea pig, adult rat, adult assayed by flow cytometry using an ATC 3000 cell sorter mouse, and adult human brain) in the presence of 1?-I- (Odam-Brucker, Wissembourg, France). The ratio of iado-1 neurotensin (0.05-0.10 nM) and various concentrations of violet/blue fluorescence was calculated for each individual protein (0.01-0.30 mg per tube). Total, nonspecific, and cell. Neurotensin was added 1 min after 1% (vol/vol) di- specific binding was measured at equilibrium by the filtration methyl sulfoxide (final concentration) or SR 48692, and the A B a 100 100 z so so

z 60 gz 40 40- z w 20 20-

0 0 0 lor 10 1° 10 9sR lo's-0(M 7 ' IO 10'11 1o 1° 10'R 4 O8 10(SM067 IO'S SR 4M2 (M) SR 42 (M1) FIG. 1. Inhibition of 'mlI-neurotensin-specific binding to adult rat brain membranes (A) and adult mouse brain membranes (B) by SR 48692. Each value is represented as the mean ± SE ofthree deter inations in the absence (o) and in the presence (g) of 10-5 M levocabastine. In some cases, the SE values were smaller than the corresponding symbols. Downloaded by guest on September 27, 2021 Pharmacology: Gully et al. Proc. Natl. Acad. Sci. USA 90 (1993) 67

Hi

I'f. -SN,, VTA

TOTA L SR 48692 1 0 b M

sR 48692 10 6 M Ni FIG. 2. SR 48692 antagonism of 1251-neurotensin labeling on coronal sections from guinea pig midbrain. 1251-Neurotensin (0.1 nM) was incubated in the absence (TOTAL) or in the presence of two concentrations of SR 48692 (10-8 and 10-6 M) or with unlabeled neurotensin (NT; 10-6 M). A light labeling is still visible in both the substantia nigra and ventral tegmental area in the presence of unlabeled neurotensin, whereas labeling has totally disappeared in the presence of the same concentration of SR 48692. SN, substantia nigra; VTA, ventral tegmental area; Hi, hippocampus. maximal neurotensin-induced increase in the fluorescence of activity in several binding assays with nonpeptide (dopa- ratio was determined. mine D1 and D2, al- and a2-adrenergic, serotonin 5-HT2, Turning Behavior in Mice. Turning behavior induced by muscarinic M1 and M2, histamine HI, and pu, 8, Kc, and o'opiate unilateral intrastriatal neurotensin injection (10 pg in 1 AL) receptors) and peptide (cholecystokinin A and B, [A8]vaso- was performed in conscious, female CD1 mice (Charles River pressin, bradykinin, neuropeptide Y, and neurokinin 1 and 2) Breeding Laboratories) according to Worms et al. (25). The ligands (data not shown). number of complete contralateral rotations was visually Autoradiographic Studies. Data presented in Fig. 2 show a recorded and accumulated over three periods of 2 min (3-5, high labeling of both substantia nigra pars compacta and 6-8, and 9-11 min) postinjection. SR 48692 (20, 40, and 80 ventral tegmental area by 1251-neurotensin in the guinea pig A.g/kg) was administered i.p. or orally (p.o.) 30 and 60 min brain. In the pars reticulata of the substantia nigra, several before intrastriatal injection, respectively. In addition, a labeled processes seemed to radiate from cells in the pars time-course study was performed with SR 48692 at 80 ,ug/kg compacta. Similar to what was observed in the rat (24)1 the (p.o.). most internal layers of the cortex, the cortical nucleus of the RESULTS Binding Studies. SR 48692 totally inhibited the specific co binding of 1251-neurotensin to homogenates from adult guinea 0 pig brain, rat mesencephalic cells, COS-7 cells transfected z 100' with the cDNA coding for the high-affinity rat brain neuro- z tensin receptor, newborn mouse brain, newborn and adult 0 human brains, and human colon carcinoma HT-29 cells. In 75 these various models, SR 48692 exhibited IC50 values (means w ± SE) ranging between 0.99 ± 0.14 nM (adult guinea pig brain I- membranes) and 30.3 ± 1.5 nM (HT-29 cell membranes), with Hill coefficients close to unity (Table 1). However, SR 48692 50. inhibited the specific binding of 1251-neurotensin in adult rat z brain and adult mouse brain with higher IC50 values (82.0 ± 7.4 and 34.8 + 8.3 nM, respectively) and lower Hill coeffi- 25 cients (0.67 ± 0.09 and 0.51 ± 0.08), which are consistent I- -10 -9 -8 with the recognition by SR 48692 of both high- and low- -7 affinity sites in the adult rat and mouse brains. In the presence [SR 48692], log M of 10 ;LM levocabastine, which recognizes only the low- affinity neurotensin sites, the IC50 obtained with SR 48692 in FIG. 3. SR 48692 antagonism ofthe stimulation by neurotensin of rat and mouse brain were much lower (5.0 ± 1.5 and 5.7 ± K+-evoked release of [3H]dopamine from striatal guinea pig slices. 1.5 Results, expressed as percent inhibition of neurotensin effect, were nM, respectively), and Hill coefficients were close to the mean ± SE from three experiments performed in triplicate. (*, unity, which indicates that this compound is more potent on P < 0.05 vs. neurotensin group; Dunnett's test). (Inset) Dose- the high-affinity than on the low-affinity binding sites in adult response curve for the neurotensin effect on 20 mM K+-stimulated murine brains (Fig. 1). The high degree of selectivity of SR release of [3H]dopamine. Dopamine release was expressed as per- 48692 for neurotensin receptors was demonstrated by its lack cent of radioactivity present in the superfusion medium. Downloaded by guest on September 27, 2021 68 Pharmacology: Gully et al. Proc. Natl. Acad. Sci. USA 90 (1993) 100' tion, autoradiograms are almost indiscernible from the film z A 0 background (Fig. 2). Stimulation of the K+-Evoked Release of [3HJDopamine from Guinea Pig Striatal Sices. Addition of 1-100 nM neu- z Z rotensin to the superfusion medium caused an enhancement -o s of the K+-stimulated (but not basal) release of [3H]dopamine 00JU -wZZ 50 (Fig. 3 Inset). The stimulatory effect (132.7% ± 15.4%; mean -JoLL ± SE) produced by 10 nM neurotensin was dose-dependently counteracted by SR 48692 (IC50 = 0.46 ± 0.02 nM; Fig. 4). Up v to 100 nM SR 48692 did not significantly affect the sponta- A A -1 { neous and K+-evoked release of [3H]dopamine by itself, -10 .9 -8 -7 -6 -5 indicating a lack of agonistic activity of the compound (data [Neurotensin], log M not shown). Ca2+ Mobilization in HT-29 Cells. As previously reported (21, 26), neurotensin induced a concentration-dependent 1.2 B 4 mobilization of Ca2+ in HT-29 cells. Increasing concentra- tions of SR 48692 produced parallel rightward shifts of the 0.8 neurotensin dose-response curve (Fig. 4A). In the course of three experiments, EC50 values for neurotensin were 4.8 ± 0.7, 6.9 ± 0.9, 11.3 ± 2.2, 21.7 ± 0.3, 58.3 ± 4.4, and 140 ± ¢_. 0.4 20 (mean ± SE in nM) in the absence and presence of 3, 10, 30, 100, and 300 nM SR 48692, respectively. Schild plot 0 analysis of the data such as those shown in Fig. 4B -7 .6 yielded -8 -6 pA2 (-log Kapp) and Ki values for SR 48692 of8.13 ± 0.03 and -0.4 7.4 ± 0.6 nM, respectively, with a slope of 0.91 ± 0.07. Turning Bebavior. The mean number of contralateral ro- [SR 48692], log M tations induced by 10 pg of intrastriatal injection of neuro- FIG. 4. Antagonism by SR 48692 of the neurote5nsin-induced tensin was 13.1 ± 1.9. These rotations were found to be calcium mobilization in HT-29 cells. (A) Concentrattion-response insensitive to spiroperidol and 6-hydroxydopamine lesions curves for neurotensin-induced calcium increase in HT--29 cells in the (data not shown). SR 48692 administered i.p. or p.o. reduced absence (a) or presence of SR 48692 at 3 (0), 10 (*), 3()(O), 100(A), the neurotensin-induced turning; 80%o antagonism was ob- and 300 nM (A). The results are expressed as the percent of the served at 80 ,ug/kg (Fig. 5 Inset). The time-course study maximal increase over the basal fluorescence ratio) induced by performed with SR 48692 at 80 j.g/kg (p.o.) revealed a neurotensin. (B) Corresponding Schild plot for the antajgonistic effect significant effect (-35%) as soon as 30 min after injection; ofSR 48692. The data are from a typical experiment. DRt-1, dose ratio maximal antagonism (-85%) was observed between 1 and 2 minus one. hr postinjection. Furthermore, SR 48692 did not reduce cholecystokinin-induced turning even at high concentrations amygdala as well as the pyramidal layer of the h ippocampal (data not shown), indicating that the antagonistic effect ofthis formation and the granular cells of the dentate g)yrus, mainly compound was specific to neurotensit. in its ventral portion, showed high densities of neurotensin binding sites. The addition ofincreasing concentrations of SR 48692 produced dramatic decreases in the deiisity of the DISCUSSION 1251-neurotensin labeling in all structures. Incubation with In the present study, we describe and characterize a potent either neurotensin or SR 48692 (1 uM) resuilted in the and selective nonpeptide neurotensin antagonist: SR 48692. complete loss of the labeling and, in contrast t:o what was This compound potently inhibited 125I-neurotensin binding in observed with unlabeled neurotensin at the sameD concentra- models in which only high-affinity neurotensin receptors are

Q 0 100. 0 I2 0 U 0 %,) z 50,

I- U- 0 w en z o0 0 0.25 as 1 2 4 6 8 12 18 24 HR

SR 48692 80 gl/Kg per Os FIG. 5. Antagonism by SR 48692 ofthe turning behavior induced by intrastriatal injection ofneurotensin (10 pg per mouse, 12 mice per group): dose-effect relationship (Inset) and time-course study after oral administration. Data are the mean + SE (Dunnett's t-test; *, P < 0.05). Downloaded by guest on September 27, 2021 Pharmacology: Gully et al. Proc. Natl. Acad. Sci. USA 90 (1993) 69 present such as newborn mouse and human brain (27, 28), 4. Rosell, S. & Rokaeus, A. (1981) Clin. Physiol. 1, 3-21. HT-29 cells (21), rat mesencephalic neurons (20), and COS-7 5. Elliott, P. J. & Nemeroff, C. B. (1986) in Neural andEndocrine cells transfected with the cloned high-affinity rat brain re- Peptides and Receptors, ed. Moody, T. W. (Plenum, New ceptor (18). SR 48692 also potently inhibited l251-neurotensin York), pp. 219-245. binding to the high-affinity binding sites in adult rat and 6. Nemeroff, C. B. (1986) Psychoneuroendocrinology 11, 15-37. mouse brain homogenates, whereas it was much less potent 7. Kitabgi, P. (1989) Neurochem. Int. 14, 111-119. 8. Ferris, C. F., Pan, J. X., Singer, E. A., Boyd, N. D., Carr- at the low-affinity, levocabastine-sensitive binding sites that away, R. E. & Leeman, S. E. (1984) Neuroendocrinology 38, are also present in these preparations (16). The IC50 of SR 145-151. 48692 for binding to adult rat brain homogenates in the 9. Alexander, M. J., Mahoney, P. D., Ferris, C. G., Carraway, presence of levocabastine was similar to that for binding to R. E. & Leeman, S. E. (1989) Endocrinology 124, 783-788. the cloned high-affinity rat brain receptor. Finally, SR 48692 10. Bissette, G., Nemeroff, C. B., Loosen, P. T., Prange, A. J., was even more potent than unlabeled neurotensin in binding Jr., & Lipton, M. A. (1976) Nature (London) 262, 607-609. to guinea pig brain tissues as shown by both radioreceptor 11. Clineschmidt, B. V., McGuffink, J. C. & Bunting, P. B. (1979) assay and autoradiographic techniques. Eur. J. Pharmacol. 54, 129-139. Consistent with the existence of neurotensin binding sites 12. Allescher, H. D. & Ahmad, S. (1991) in Neuropeptide Function on terminals of the nigrostriatal dopaminergic pathway (29, in the Gastrointestinal Tract, ed. Daniel, E. E. (CRC, Boca Raton, FL), pp. 309-400. 30), neurotensin was shown to promote an increase in K+- 13. Rioux, F., Kerouac, R., Quirion, R. & St.-Pierre, S. (1982) evoked release of [3H]dopamine from rat, cat, and rabbit Ann. N. Y. Acad. Sci. 400, 56-74. striatal slices (31-33). This observation was extended here to 14. Kitabgi, P., Checler, F., Mazella, J. & Vincent, J. P. (1985) guinea pig striatal slices. SR 48692 was without effect on Rev. Clin. Basic Pharmacol. 5, 397-486. basal and K+-evoked dopamine release, but completely 15. Schotte, A., Leysen, J. E. & Laduron, P. M. (1986) Naunyn- antagonized the effect of neurotensin on K+-stimulated do- Schmiedeberg's Arch. Pharmacol. 333, 400-405. pamine release with a potency similar to its affinity for guinea 16. Kitabgi, P., Rostene, W., Dussaillant, M., Schotte, A., Ladu- pig brain receptors. ron, P. & Vincent, J. P. (1987) Eur. J. Pharmacol. 140, 285- Neurotensin has previously been reported to stimulate 293. 17. Kostka, P. (1991) in Neuropeptide Function in the Gastroin- Ca2+ mobilization through an increase in inositol phosphates testinal Tract, ed. Daniel, E. E. (CRC, Boca Raton, FL), pp. in the human colon carcinoma HT-29 cell line (21, 26). 249-271. Binding experiments and intracellular Ca2+ measurements 18. Tanaka, K., Masu, M. & Nakanishi, S. (1990) Neuron 4, showed that SR 48692 acted as a potent and full competitive 847-854. antagonist of the neurotensin-induced Ca2+ response in this 19. Sadoul, J. L., Mazella, J., Amar, S., Kitabgi, P. & Vincent, system. J. P. (1984) Biochem. Biophys. Res. Commun. 120, 812-819. Evidence suggests that neurotensin receptors located 20. Dana, C., Pelaprat, P., Vial, M., Brouard, A., Lhiaubet, A. M. postsynaptically to, rather than on, dopamine neurons may & Rostene, W. (1991) Dev. Brain Res. 61, 259-264. be involved in some behavioral changes produced by central 21. Bozou, J. C., Rochet, N., Magnaldo, I., Vincent, J. P. & Kitabgi, P. (1989) Biochem. J. 264, 871-878. injection of neurotensin (34, 35). In a well-defined cholecys- 22. Luthman, H. & Magnusson, G. (1983) Nucleic Acids Res. 11, tokinin model (25), we found that unilateral injection of 1295-1308. neurotensin into the striatum of unrestrained mice produced 23. Lugrin, D., Vecchini, F., Doulut, S., Rodriguez, M., Martinez, dopamine-independent circling behavior. SR 48692 (80 ,&g/kg J. & Kitabgi, P. (1991) Eur. J. Pharmacol. 205, 191-198. i.p. or p.o.) was able to antagonize this effect ofneurotensin. 24. Moyse, E., Rostene, W., Vial, M., Leonard, K., Mazella, J., These data demonstrate that SR 48692 has a good oral Kitabgi, P., Vincent, J. P. & Beaudet, A. (1987) Neuroscience bioavailability and strongly support the conclusion that the 22, 525-536. antagonist can cross the blood-brain barrier. 25. Worms, P., Gueudet, C. & Biziere, K. (1986) Life Sci. 39, In summary SR 48692, a nonpeptide antagonist of neuro- 2199-2208. 26. Turner, J., James-Kracke, M. R. & Camden, J. M. (1990) J. tensin receptor may be very useful in experiments to design Pharmacol. Exp. Ther. 253, 1049-1056. the physiological and putative pathological roles of neuro- 27. Mazella, J., Chabry, J., Zsurger, N. & Vincent, J. P. (1989) J. tensin. Biol. Chem. 264, 5559-5563. 28. Zsurger, N., Chabry, J., Coquerel, A. & Vincent, J. P. (1992) We thank Nicole Zsurger and Antoine Coquerel for the gift of Brain Res. 586, 303-310. newborn human brain tissue, Shigetada Nakanishi for the gift of the 29. Quirion, R., Chiueh, C. C., Everist, H. D. & Pert, A. (1985) rat brain neurotensin receptor cDNA, Dariush Farahifar for his help Brain Res. 327, 385-389. in cytometry experiments, Micheline Vial for her help in autoradio- 30. Herve, D., Tassin, J. P., Studler, J. M., Dana, C., Kitabgi, P., graphic studies, and Anne-Marie Lhiaubet for the radiolabeled ligand Vincent, J. P., Glowinski, J. & Rostene, W. (1986) Proc. NatI. preparation. Acad. Sci. USA 83, 6203-6207. 31. De Quidt, M. E. & Emson, P. C. (1983) Brain Res. 274, 1. Carraway, R. E. & Leeman, S. E. (1973) J. Biol. Chem. 248, 376-380. 6854-6861. 32. Battaini, F., Govoni, S., Di Giovine, S. & Trabuchi, M. (1986) 2. Carraway, R. E. & Leeman, S. E. (1976) J. Biol. Chem. 251, Naunyn-Schmiedeberg's Arch. Pharmacol. 332, 267-270. 7045-7052. 33. Markstein, R. & Emson, P. (1988) Eur. J. Pharmacol. 152, 3. Emson, P. C., Goedert, M. & Mantyh, P. W. (1985) in Hand- 147-152. book of Chemical Neuroanatomy: Vol. 4, GABA and Neu- 34. Kasckow, J. & Nemeroff, C. B. (1991) Regul. Pept. 36, 153- ropeptides in the CNS, Pt. 1, eds. Bjorklund, A. & Hokfelt, T. 164. (Elsevier, Amsterdam), pp. 355-405. 35. Rivest, R. & Masden, C. A. (1992) Neuroscience 47, 341-349. Downloaded by guest on September 27, 2021