Chapter 18 Tachykinin receptors in the CNS

E.R. de K l a t , V.M.Wiegant and D. de Wied (Eds.)

Progrcrr in Brain Research. Vol. 12

0 1987 Elsevier Science Publishers B.V. (Biomedical Division)

197

CHAPTER 18

Tachykinin receptors in the CNS J. Glowinski", Y. Torrens", M. SaEroy", L. Bergstr(lma,J. C. Beaujouan", S. Lavielleb, 0. Plouxb, G. Chassaingb and A. Marquetb "Chaire de Neuropharmacologie.INSERM U.114, Colkge de France, 11 place Marcelin Berthelot. 75231 Paris Cedex 5 and bL.aboratoire Chimie Organique Biologique, EA 455, Universitk Paris 6,place Jussieu. 75005 Paris. France

Introduction Apart from substance P (SP),other tachykinins are present in the CNS of mammals. NeurokininA (NKA, also known as neuromedin L or substance K) and neurokinin B (NKB, also known as neuromedin K) have both been identified in the brain and spinal cord of vertebrates (Kimura et al., 1983 ;Minamino et al., 1984). In addition, gene cloning techniques have allowed the determination of the nucleotide sequence of two different SP precursors in the bovine caudate nucleus (Nawa et al., 1983). Besides SP, NKA, a peptide structurally related to kassinin was found in one of these precursors. As was done for opioid receptors, bioassays on peripheral organs and the recent availability of some tachykinin antagonists have facilitated the identification of several classes of peripheral tachykinin receptors (Regoli et al., 1985). Binding studies with appropriate ligands have confirmed the heterogeneity of tachykinin receptors in peripheral tissues (Buck and Burcher, 1986; Buck et al., 1984). It is now generally agreed, on the basis of binding studies, that there occur two classes of tachykinin receptors in the brain with substance P and NKB as endogenous ligands. Starting in 1980, we investigated the central tachykinin receptors ;this research was prompted by investigations made on striato-nigral SP neurons. Following the demonstration of the in

vivo release of SP in the cat substantia nigra (Michelot et al., 1979), we had observed that striato-nigral SP neurons were involved in the regulation of nigro-striatal dopaminergic neurons (Glowinski et al., 1980). Some aspects of our research on central SP receptors will be summarized briefly.

Kinetic and pharmacological properties of lZ5IBolton Hunter SP and '251-Bolton Hunter eledoisin binding sites on rat brain synaptosomes Crude synaptosomes from the rat brain (or from defined brain structures) were used since satisfactory results had been obtained with intact cells, i.e. closed systems from the periphery by other groups (Liang and Cascieri, 1981; Sjodin et al., 1980) and from the brain in our laboratory (Beaujouan et al., 1982). Initial studies were performed with *2SI-BoltonHunter SP ( I2'I-BHSP), a ligand which exhibits a biological activity similar to that of SP on the guinea-pig ileum (Michelot et al., 1980). We then synthesized a 1251-Bolton Hunter derivative of eledoisin ( 12'I-BHE) to look for the existence of a second type of central tachykinin receptor (Beaujouan et al., 1984). Eledoisin was described as being more potent than SP in contracting the hamster urinary bladder or the rat vas deferens (Erspamer, 1981) or in inducing scratching (Share and Rackham, 1981) and drinking behavior (De Car0 et al.,

198

1980) when injected into certain brain structures. Synaptosomes from the whole brain (minus the cerebellum) and from the cerebral cortex were used for the initial studies on 12'I-BHSP and 12'I-BHE binding respectively. Specific binding of 12'I-BHSP and of 12'1BHE could be demonstrated, non-specific binding being neghgible or very low (Beaujouan et al., 1984; Viger et al., 1983). The specific binding was temperature-dependent, saturable and reversible. In addition, ouabain (10- mol/l) failed to reduce 1251-BHSPor 12'I-BHE binding, indicating that these ligands were not transported into synaptosomes by a high affinity uptake process. Scatchard and Hill plots from equilibrium studies indicated the existence of a single class of noninteracting binding sites both with 1251-BHSPand IZ51-BHE.However, results obtained with cortical synaptosomes revealed marked differences in the Kd (1.1 nmol/l and 15 nmol/l) and B,, (14 and 297 fmol/mg protein) values for "'I-BHSP and 12'I-BHE binding respectively. Competition experiments with several tachykinins, SP C-terminal fragments, SP analogs or SP antagonists indicated that there were also differences in the pharmacological properties of 12'I-BHSP and 12'I-BHE binding sites (Table 1). Unlike SP, eledoisin, kassinin, NKA or NKB were only weak inhibitors of L251-BHSPspecific binding, physalaemin being slightly less potent than SP. In contrast kassinin and NKB were the most potent of all tachykinins at inhibiting I2'I-BHE binding while SP was the least potent. Thus, we suggested that NKB was the endogenous ligand for 12'I-BHE binding sites (Torrens et al., 1984). SP was much more potent than all its C-terminal fragments at inhibiting '"I-BHSP binding. A relationship was found between the length of SP C-terminal fragments and their affinity for 12'1BHSP binding sites, longer fragments being the most potent (Viger et al., 1983). In contrast, some shorter SP C-terminal fragments, SP(6-11) as well as (p.Glu6)SP(6-1 1) particularly, inhibited 12TBHEbinding with a greater potency than SP,

and these SP C-terminal fragments had an affinity for I2'I-BHE binding sites comparable to that of eledoisin itself (Torrens et al., 1985). Similar observations with (p.Glu6)SP(6-1 1) were made by Cascieri and Liang (1984). SP N-terminal fragments were without activity on either 12'1BHSP or I2%BHE specific binding. According to Watson et al. (1983), SP-methyl ester has a much higher affinity for SP receptors in peripheral organs than for other types of tachykinin receptors. Data obtained with cortical synaptosomes agree with this observation, since SP-methyl ester was relatively potent in inhibiting '"I-BHSP binding whilst it did not affect 12'I-BHE binding. Unlike SP methyl ester, DiMeC-7, a metabolically stable SP analog had little affinity for cortical '"I-BHSP binding sites whilst it was as potent as SP at inhibiting 12'I-BHE specific binding (Torrens et al., 1985).

TABLE 1 Inhibitory effects of tachykinins, SP analogs and SP antagonists on lz5I-BHSP and '251-BHEspecific binding on rat cortical synaptosomes" Compound SP Physalaemin NKA Eledoisin Kassinin NKB

6.0 x lo-'' 3.2 x 1.2 x 10-7 1.4 x lo-' 2.5 x 1 0 - 6 1.2 x 1 0 - 6

1.3 x 5.6 x l o - ' 1.0 x 10-7 1.4 x lo-' 5.9 x 1 0 - ~ 5.1 x 10-9

SP(6-11) SP(7-11)

5.2 x 10-7 > 10-5

3.9 x 1 0 - 8 2 . 0 ~1 0 - 6

SP( 1-1 1)free acid SP( I-9)amide

7.1 x > 10-5

> 10-5 > 10-5

SP methyl ester DiMeC-7

1.0 x 10-7 7.0 1 0 - 6

(D-A~~',D-T~~~",L~~'')SP 4.9 x (~-Pro~,D-Trp~*~.'')SP(4-11)2.7 X

> 10-5

1.5 x 1 0 - ~

> 10-5 1.2 X

" Data represent means of results obtained from 3 to 10 experiments.

199

Several antagonists of tachykinins are available, some of them being potent at the periphery. Antagonists of the octapeptide and undecapeptide series synthesized respectively by Regoli's group (Regoli et al., 1984) and Rosell and coworkers (Rosell and Folkers, 1982) slightly inhibited 12%BHSP specific binding on cortical synaptosomes (D-Arg', D-T~P'.~,Leu' ')SP being the most potent (Ki 4.9 x 10-7mol/l). In contrast, these compounds had no or negligible effects on '251-BHE binding (Torrens et al., 1985). Recently, the effects of several alkylating agents on the binding of 12'I-BHSP and '251-BHE were examined using cortical synaptosomes from the rat brain. Preliminary data indicate that alkylating agents such as p-chloromercuriphenylsulfonic acid, $5 ' -dithiobis(2-nitro)benzoic acid and N-ethylmaleimide reacting with thiol groups decreased IZ5I-BHEbinding but did not affect that of 1251-BHSP.This provided further evidence for marked differences in the structure of the two types of binding sites.

DifTerences in the regional distribution of lZ5IBHSP and '*'I-BHE binding sites Striking differences were seen in the regional distribution of '251-BHSP and '"I-BHE binding sites when binding studies were done with crude synaptosomal preparations from different brain structures (Beaujouan et al., 1984). 12%BHSP binding was particularly elevated in the septum while negligible binding of 1251-BHEwas found in this structure. Contrasting with these results, the cerebral cortex contained the highest I2'I-BHE specific binding while '251-BHSP binding was moderate. Although substantial amounts of '251-BHSPand 1251-BHEbinding sites occurred in the hypothalamus, the hippocampus and the striatum, both the cerebellum and the substantia nigra were poorly labelled with both ligands. The lack of '251-BHSPbinding sites in the substantia nigra was surprising,consideringthe high levels of SP found in this structure.

Several groups have performed autoradiographic studies on brain sections using either 3H-SP or '251-BHSP(Quirion et al., 1983; Shults et al., 1984). The results obtained can be compared with those from our own study undertaken in order to distinguish the precise localization of '*'I-BHSP and I2%BHE binding sites (Beaujouan et al., 1986). 12%BHSP binding was seen particularly in more superficial layers of the neocortex (I, 11, 111). High levels of '251-BHSP binding were found in: nuclei of the septum (diagonal band of Broca, septohippocampal nucleus, dorsal part of the lateral septal nucleus), the rostro-dorsal part of the hippocampus and discrete nuclei such as the endopynform nucleus, the anterior cortical amygdaloid nucleus, the vermis columns (9-lo), the dorsal tegmental nucleus, the hypoglossal and ambiguus nucleus. All these structures were labelled only weakly by '251-BHE. In contrast, layers IV andV of the cerebral cortex and the paraventricular and supraoptic hypothalamic nuclei were densely labelled by I2'I-BHE but not by '"1-BHSP. Marked differences were also seen in the spinal cord (Ninkovic et al., 1984). 1251-BHSPbinding sites were localized in high concentrations in layers I and I1 (substantia gelatinosa) of the dorsal horn, around the central canal (lamina X)and in association with motor neurons in the lateral ventral horn. The localization of 12'I-BHE binding sites was restricted to layer I and to the outer part of layer I1 (110) of the dorsal horn but '251-BHE binding sites were not found associated with motor neurons in the ventral horn.

Similarity between the properties of '"I-BHE and 3H-NKB binding sites Our data on the pharmacologicalproperties of the '251-BHSP binding sites obtained either with embryonic intact mesencephalic cells from the mouse (see below) or synaptosomes from the rat brain have been confirmed by other workers who used either 3H-SP or 1251-BHSP and brain membranes (Cascieri and Liang, 1983; Quirion

200

and Pilapil, 1984). In fact, similar results were also obtained when we compared the pharmacological properties of 'H-SP and 9 - B H S P binding sites on brain synaptosomes. All these studies allow the conclusion that the endogenous ligand for high affinity lZ5I-BHSPbinding sites is indeed SP. As discussed already, we have suggested that NKB was the endogenous ligand for '"I-BHE binding sites. In order to confirm this hypothesis, 'H-NKB with a high specific activity (75 Ci/mmol) was synthesized (Chassaing et al., 1985) and its binding was studied on crude synaptosomes from the cerebral cortex (Bergstrdm et al., 1987). Specific binding of 'H-NKB, which was temperature-dependent, saturable and reversible could be demonstrated. Scatchard analysis and Hill plots revealed the existence of a single population of non-interacting sites (& = 4.3 nmol/l, B,,, 123 fmol/mg protein). Competition studies with several tachykinins, SP(6-1 l), DiMeC-7, SP methyl ester indicated that the pharmacological properties of 'H-NKB and '"1-BHE binding sites were identical. Indeed, NKB was found the most potent at inhibiting 'H-NKB binding (NKB > eledoisin > kassinin > physalaemin > NKA > SP). In addition, the 12'I-BH derivative of N U had a higher affinity than NKA for 3H-NKB, therefore, lZ5I-BHNKA may not label specifically NKA binding sites in the brain as suggested by Quirion and Dam (1985). Finally, the similarity of 'H-NKB and 12'I-BHE binding sites was confirmed further by autoradiographic analysis on rat brain sections since a strict superposition in the localization of 'H-NKB and lZ5I-BHEbinding sites was observed.

Cellular localization of 1251-BHSPbinding sites Primary cultures allow the precise cellular localization of receptors to be defined. In fact, our initial study on central SP receptors was performed on intact mesencephalic cells from the mouse embryo grown for five days in primary

culture (Beaujouan et al., 1982). Specific binding sites for 12sI-BHSP were found under culture conditions allowing the development of both neuronal and glial cells (medium enriched with foetal calf serum) or of neuronal cells alone (synthetic medium without serum). Kinetic and pharmacological characteristics of '"I-BHSP binding sites were similar to those described previously with rat brain synaptosomes. However, a higher affinity(& = 0.17nM) of the binding sites was found. This could result from hypersensibility of SP receptors due to the lack of contacts between presynaptic SP fibers and their target cells. Therefore, high affinity specific "%BHSP binding sites are already expressed on embryonic neuronal cells and this was confirmed by autoradiography. Differences in the amount of ",IBHSP specific binding sites were found in different brain structures, but mesencephalic cells had the highest number of lZSI-BHSPbinding sites. Interestingly, other authors who used 'H-SP and dissociated cells from the rat brain grown in primary cultures have reached similar conclusions concerning the kinetic and pharmacological properties of SP receptors (RUegg, 1983). Using biochemical experiments and autoradiographic analysis, we recently demonstrated the occurrence of "'I-BHSP binding sites on astrocytes prepared from several structures of the mouse brain (Torrens et al., 1986).Three- or fourweek-old confluent glial cells in primary culture originating from one-day-old newborn mice were used. The kinetic and pharmacological characteristics of the high affinity lZ5I-BHSPbinding sites were similar to those found on synaptosomes. In addition, SP was found to stimulate phosphatidylinositol breakdown in a dose-dependent fashion. Although the ED,, value for the SP-evoked response was identical to the Kd for 12'I-BHSP high affinity binding, further experiments are required to determine whether or not the receptors involved in the biological response exhibit pharmacological properties identical to those of I2,I-BHSP binding sites. Interestingly, neither 1251-BHSPbinding sites nor a SP-induced

20 1

formation of inositol phosphates could be fount when three to four-week-old cultures of astrocyte from newborn rats were used. The functional rol of SP receptors on astrocytes from the mous brain still has to be elucidated. However, likother receptors for amino-acids, amines and neuropeptides which have been identified on astrocytes during the last few years, these SP receptors could contribute to neuronal-&a interactions. Structural requirements for interaction with '*'IBHSP binding sites Three complementary types of approach were used in order to get a better knowledge of the structural requirements for the 12%BHSP binding sites: (1) determination of the conformation of SP in various solvents; (2)analysis of the influence of SP amino-acids on both the binding potency and the conformation; (3) finally, the synthesis of constrained cyclic analogs of SP simulating the three-dimensional structure of SP in solution. NMR studies indicated that the conformation of SP was strongly influenced by its environment. There was an extended conformation of SP in dimethylsulfoxide and pyridine. In water, SP presented a complex conformational equilibrium. The addition of sodium dodecylsulfate induced a preferential a-helical structure similar to that observed in methanol. Since Lembeck et al. (1979) have shown that SP binds to phosphatidylserine and phosphatidylethanolatnine,we suggested that SP might bind to its receptor via a two-step binding mechanism: the membrane inducing a specific conformation of SP similar to that observed in methanol. The main features of the conformational model proposed are the flexibility of the N-terminal Arg-Pro-Lys, the a-helical structure of Pro4Phe8and the interaction of the C-terminal carboxamide with the primary amide from both glutamines (Chassaing et al., 1986). Physalaemin, another tachykinin with a pharmacological profile similar to that of SP,

presented the same a-helical conformation for the core of the peptide in methanol whereas NKA, a weak agonist of the '"I-BHSP specific binding sites, exhibited a completely different threedimensional structure. Several SP analogues were synthesized to deternliae the importance of each amino-acid of SP for its binding to '"I-BHSP binding sites. For comparison, the potency of these analogues was also estimated on 12%BHE binding (cortical synaptosomes) and on the guinea-pig ileum which contains three sub-types of tachykinin receptors. From the data obtained and assuming that the core of SP exists in an a-helicoidal structure, three remarks could be made (Lavieile et al., 1986): (1) the SP specific binding site probably recognizes the side of the helix bearing the two sidechains of the Phe' and Phe8; (2)the arginine guanidinium interacts with either a carboxylate or a phosphate function of the binding site; (3) finally, since the hydrogen bond interaction between the C-terminal Met1'-NH2 and the Glu5 residue was no longer present, as determined by NMR, in the (Met6)SP analog, the C-terminal Gly-Leu-Met-NH, undergoes a conformation change. This translocation allows the interactions of the C-terminal amide with a carboxylate and that of the sulfur atom with an electrophile of the binding site. The question arises as to whether the threedimensional structure observed for SP in methanol had any relevance to the conformation interacting with the binding site. In order to resolve this problem, we have synthesized constrained cyclic analogues of SP, simulating the conformation of SP. Thus, the efficiencies of two groups of cyclic analogues of SP were studied (Table 2). The disulfide bridge constraints were designed on the basis of our conformational studies of SP a@ physalaemin, indicating an a-helical structure for the core of these two tachykinins (group I) or on the basis of studies reported in the literature suggesting a turn in the C-terminal sequence (group 11). Only (D-cyS3,cyS6)sP, simulating the a-helix, presented some substantial

202

TABLE 2 Effects of SP cyclic analogs on guinea-pig ileum, on l2%BHSP specific binding to rat brain synaptosomes and on '251-BHE specific binding to rat cortical synaptosomes Peptides

Guinea-pig ileum ECso (M)

l2%BHSP

'251-BHE

R.A.

IC50 (MI

R.A.

IC5, (W

R.A.

50 2 3

2.0 x 10-8 1.7 x 2.0 x 10-7

3 0.03 0.3

7.2 x lo-' > 10-5 1.4 x 10-6

-

9.2 x 5.3 x 1.3 x 3.6 x 2.7 x

0.07 0.12 0.05 0.18 0.24

> 10-5 > 10-5 > 10-5 > 10-5 > 10-5

-

Group I (D-cyS3,cyS6)sP ( D-cyS4,cyS7)sP (D-cySs,c~Ss)sP

4 x 10-9 I x 10-7 7 x 10-8

Group I1 (Cys5,Cys~)SP (Hcy5,Hcyg)SP (~-cys~,Hcy")SP (Cys5,Cys' ' )SP (HcyS,Hcy' ' )SP

I x 10-7 x 10-7 x 10-6

2 2 4 3.3

x 10-7 x 10-7

2 2 0.1 0.5 0.6

10-7 10-7 10-6 10-7 10-7

7

0.4

Data represent means of results obtained from 3 to 5 independent experiments (R.A., reletive affinity when compared to SP, 100).

potencies as it was as active as SP in the guineapig ileum bioassay and its IC,, for the '251-BHSP specific binding sites was 20 nmol/l. In addition ( ~ - c y s ~ , C y s ~ )was S P as potent in inhibiting the lZSI-BHEspecific binding. These findings led us to propose that these two types of tachykinin binding sites may recognize a similar threedimensional structure of the core of the tachykinins. The very weak potencies of the peptides from group I1 suggest that a certain degree of flexibility in the C-terminal sequence is required. References Beaujouan, J.C., Torrens, Y.,Herbet, A., Daguet, M.C., Glowinski, J. and Prochiantz, A. (1982) Specific binding of an immunoreactive and biologically active '251-labelled substance P derivative to mouse mesencephalic cells in primary culture. Mol. Pharmacol., 22: 48-55. Beaujouan, J.C., Torrens, Y.,Viger,,A. and Glowinski, J. (1984) A new type of tachykinin binding site in the rat brain characterized by specific binding of a labeled eledoisin derivative. Mol. Pharmacol., 26: 248-254. Beaujouan, J.C., Torrens, Y.,Saffroy, M. and Glowinski, J. (1986) Quantitative autoradiographic analysis ofthe distribution of binding sites for '251-Bolton Hunter derivatives

of eledoisin and substance P in the rat brain. Neuroscience, 18: 857-875.

Bergstrbm, L., Torrens, Y., Saffroy, M., Beaujouan, J.C., Lavielle, S., Chassaing, G., Morgat, J. L., Glowinski, J. and Marquet, A. (1987) 'H-Neurokinin B and '251-Bolton Hunter eledoisin label identical tachykinin binding sites in the rat brain. J. Neurochem., 48: 125-133. Buck, S . H. and Burcher, E. (1986) The tachykinins: a family of peptides with a brood of 'receptors'. TIPS, 7: 65-68. Buck, S. H., Burcher, E., Shults, C.W., Lowenberg, W.and ODonohue,T. L. (1984) Novel pharmacology of substance K-binding sites: a third type of tachykinin receptor. Science, 226: 987-989. Cascieri, M. A. and Liang, T. (1983) Characterization of the substance P receptor in rat brain cortex membranes and the inhibition of radioligand binding by guanine nucleotides. J . Biol. Chem., 258: 5158-5164. Cascieri, M. A. and Liang, T. (1984) Binding of ( 1251)Bolton Hunter conjugated eledoisin to rat brain cortex membranes. Evidence for two classes of tachykinin receptors in the mammalian central nervous system. Life Sci., 35: 179-184.

Chassaing, G., Lavielle, S., Marquet, A,, Genet, R., Fromageot, P. and Morgat, J.L. (1985) A new procedure for 'H-labelling of the methionine-containing peptides: application to the labelling of methionine-amide, substance P, neurokinin A and B. In: C. M. Deber, and K. D. Kopple (Eds.), Proceedings of the 9th American Peptide Symposium. Toronto.

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Chassaing, G., Convert, 0. and Lavielle, S.(1986) Preferential conformation of S P in solution. Eur. J. Biochem., 154: 77-85.

De Caro, G., Massi, M. and Micossi, L.G. (1980) Modifications of drinking behavior and of arterial blood pressure induced by tachykinins in rats and pigeons. Psychopharmacology, 68: 243-247. Erspamer, V. (1981) The tachykinin peptide family. TINS, 4: 267-269.

Glowinski, J., Michelot, R. and Cheramy, A. (1980) Role of striato-nigral substance P in the regulation of the activity of the nigro-striatal dopaminergic neurons. In: E. Costa and M. Trabucchi (Eds.), Neural peptides and neuronal communication, Raven Press, New York, pp. 51-61. Kimura, S., Okada, M., Sugita, Y., Kanazawa, I. and Munekata, E. (1983) Novel neuropeptides, neurokinin A and B, isolated from porcine spinal cord. Proc. Jpn. Acad. Sci.. 56: 101-104. Lavielle, S., Chassaing, G., Julien, S.,Besseyre, J., Marquet, A., Beaujouan, J. C., Torrens, Y. and Glowinski, J. (1986) Influence of the amino acids of substance P in the recognition ofits receptor: affinities of synthesized SP analogues for the specific '251-BHSP binding site on rat brain synaptosomes. Neuropeptides, 7: 191-200. Lembeck, F., Saria, A. and Mayer, N. (1979) Substance P: model studies of its binding to phospholipids. Naunyn Schmiedeberg's Arch. Pharmacol., 306: 189-194. Liang, T. and Cascieri, M.A. (1981) Substance P receptors on parotid cell membranes. J. Neurosci., 1: 1133-1141. Michelot, R., Leviel, V., Torrens, Y., Glowinski, J. and Cheramy, A. (1 979)In vivo release of substance P in the cat substantia nigra. Neurosci. L e f f . ,15: 141-146. Michelot, R.. Gozlan, H., Beaujouan, J.C., Besson, M.J., Torrens, Y. and Glowinski, J. (1980) Synthesis and biological activities of substance P iodinated derivatives. BBRC, 95: 491-498.

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Nawa, H., Hirose, T., Takashima, H., Inayama, S. and Nakanishi. S. (1983) Nucleotide sequences of cloned cDNAs for two types of bovine brain substance P precursor. Nature, 306: 32-26. Ninkovic, M., Beaujouan, J.C., Torrens, Y., Saffroy, M., Hall, M. D. and Glowinski, J. (1984) Differential localization of tachykinin receptors in rat spinal cord. Eur. J. Pharmacol., 106: 463-464. Quirion, R. and Dam, T. V. (1985) Multiple tachykinin recep-

tors in guinea pig brain. High densities of substance K (neurokinin A) binding sites in the substantia nigra. Neuropeptides, 6: 191-204. Quirion, R. and Pilapil, C. (1984) Comparative potencies of substance P, substance K and neuromedin K on brain substance P receptors. Neuropeptides, 4: 325-329. Quirion, R., Shults, C. W., Moody, T. W., Pert, C.B., Chase, T. N. and ODonohue, T. L. (1983) Autoradiographic distribution of substance P receptors in rat central nervous system. Nature, 303: 714-717. Regoli, D. (1985) Peptides antagonists, TIPS, 6: 481-484. Regoli, D., DOrleans-Juste, P., Escher, E. and Mizrahi, J. (1984) Receptors for substance P. I. The pharmacological preparations. Eur. J . Pharmacol., 97: 161-170. Rosell, S . and Folkers, K. (1982) Substance P-antagonists: a new type of pharmacological tool. TIPS, 3: 21 1-212. RUegg, V. T. (1983) A binding site for substance P on dissociated cells from cultures of rat CNS. In: P. Skrabanek and D. Powell (Eds.), Substance P. Dublin, pp. 65-66. Share, N.N. and Rackham, A. (1981) Intracerebral substance P in mice: behavioral effects and narcotic agents. Brain Res., 211: 379-386. Shults, C.W., Quirion, R., Chronwall, B., Chase, N. N. and ODonohue, T. L. (1984) A comparison of the anatomical distribution of substance P and substance P receptors in the rat central nervous system. Peptides, 5: 1097-1 128. Sjodin, L., Brodin, E., Nilsson, G. and Coulon, T. P. (1980) Interaction of substance P with dispersed pancreatic acinar cells from the guinea pig: binding of radioiodinated peptides. Acta Physiol. Scand., 109: 97-105. Torrens, Y., Lavielle, S., Chassaing, G., Marquet, A., Glowinski, J. and Beaujouan, J. C. (1984) Neuromedin K, a tool to further distinguish two central tachykinin binding sites. Eur. J . Pharmacol., 102: 381-382. Torrens, Y., Beaujouan, J.C. and Glowinski, J. (1985) Pharmacological characterization of two tachykinin binding sites in the rat cerebral cortex. Neuropeptides, 6: 59-70. Torrens, Y., Beaujouan, J.C., Saffroy, M., Daguet de Montety, M.C., Bergstrt)m, L. and Glowinski, J. (1986) Substance P receptors in primary cultures of cortical astrocytes from the mouse. PNAS, 83: 9216-9220. Viger, A., Beaujouan, J.C., Torrens, Y. and Glowinski, J. (1983) Specific binding of a '251-substance P derivative to rat brain synaptosomes. J. Neurochem., 40: 1030-1038. Watson, S.P., Sandberg, B. E. B., Hanley, M.R. and Iversen, L. L. (1983) Tissue selectivity of substance P alkyl esters suggesting multiple receptors. Eur. J. Pharmacol., 87: 77-84.