Neuropeptides 6: 191-204, 1985

MULTIPLE TACHYKININ RECEPTORS IN GUINEA PIG BRAIN. HIGH DENSITIES OF SUBSTANCE

K (NEuROKININ A) BINDING SITES IN THE SUBSTANT~A NIGRA,

Remi Quirion and Than-Vinh Dam. Douglas Hospital Research Centre and Dept, of

Psychiatry, Faculty of Medicine, McGill University, 6875 LaSalle Blvd., Verdun,

Quebec, (Canada) H4H lR3. (reprint requests to R-Q.)

ABSTRACT

Receptor binding characteristics of labelled substance P (SP) and subs-

tance K (SK) were investigated in guinea pig membrane preparations and by in

vitro receptor autoradiography. The two ligands bind to single class of sites

withKd of 2.1 nM and Bmax of 37.0 fmol/mg protein for SP and IO.6 nM and 50.3

fmol/mg protein for SK. Ligand selectivity patterns markedly differ for the

two 1 igands. On SP binding sites, SP > physalaemin > eledoisin > SK > kassin-

in > Neuromedin K, On the other hand, SK > kassinin > Neuromedin K > eledois-

in > SP > physalaemin on SK binding sites. The autoradiographic distribution

of SP and SK binding sites is also different. While the cerebral cortex, substantia nigra and cerebellum contain very low to low densities of SP sites,

they are all rich in SK binding sites, These data clearly demonstrate the

existence of at least two classes of tachykinin binding sites in guinea pig

brain. Moreover, the high density of SK binding sites present in the subs-

tantia nigra suggests possible physiological roles for SK in this region,

INTRODUCTION

Recently, various groups have demonstrated the existence of a family of

mammalian tachykinins, including substance P (l), substance K (neurokinin A)

(2,3), neuromedin K (neurokinin B) (2,4) and possibly a physalaemin-related

peptide (5). Thus far, the isolation of two tachykinin precursors has been

reported (6) o a-preprotachykinin contains only one copy of substance P while

f3-preprotachykinin contains one copy each of substance P and substance K,

Finally, recent immunohistochemical data indicate that both substance P and

substance K are similarly distributed in rat brain (7).

Much evidence suggests that the various tachykinins could act on dif-

ferent classes of receptors (8-12). Even before the discovery of multiple mammalian tachykinins, various groups proposed the existence of sub-types of tachykinin receptors, mainly on the basis of data obtained in bioassays (8-12).

More specifically, Lee et al (9) suggested the existence of two substance P receptors o On the substance P-P sub-type, substance P, physalaemin, eledoisin

and kassinin are almost equipotent while the last two are much more active than substance P on the substance P-E receptor, MO reove r , recent data have shown

191

that the two new neurokinins, substance K and neuromedin K are mOre potent on

the substance P-E than the substance P-P receptor sub-type (13-15). Finally, receptor binding data also support the existence of multiple tachykinin receptor sub-types (12,16-21) and it has been shown that substance P (12,22-26), eledoisin (27) and substance K (12,28) binding sites are differently distribu- ted in rat brain, We now report that substance P and substance K binding sites possess different ligand selectivity pattern and autoradiographic distributions in guinea pig brain. The high density of substance K binding sites in the substantia nigra suggests that this tachykinin may play an important role in striato-nigral interactions.

MATERIALS AND METHODS

For membrane binding, whole brains (minus cerebelli) were removed on ice

from 500 g male guinea pigs (Charles River, Canada). Membranes were prepared as follows: Brains were homogenized in 10 volumes of 150 mM Tris.HCl buffer, pH 7.4 at 4'C plus 120 mM NaCl and 5 mM KCI using a Brinkmann polytron

(setting 6, 20 set). The homogenate was then centrifuged for 10 min at

50,000 go The supernatant was discarded and the pellet resuspended in 50 mM Tris.HCl buffer, pti 7.4 at 4'C plus 300 mM KC1 and 10 mM EDTA,K2 before in- cubation on ice for 30 min with gentle agitation. After centrifugation as above, the pellet was resuspended in 20 volumes of 50 mM Tris.HCl buffer,

pH 7.4 at 4'C, recentrifuged at 50,000 g and the supernatant discarded. The pellet was washed twice with 50 mM Tris.HCl buffer, pH 7.4 at 4'C before final resuspension in 60 volumes of the same buffer. Aliquots were taken for protein determination (29).

For binding assays 200 ~1 of membrane suspension were incubated for 90 min at 25'C with 200 ~1 of 50 mM Tris.HCl buffer, pH 7.4 at room temperature containing 3 mM MnC12, 0,02% bovine serum albumin, 2 ug/ml chymostatin (Sigma), 4 ug/ml leupeptin (Sigma), 40 ug/ml bacitracin (Sigma), and either [12'1]

Bolton-Hunter substance P (2200 Ci/mmol) or [ lz51] Bolton-Hunter substance K (2200 Ci/mmol) and various other compounds as indicated. incubations were terminated by rapid filtration (Cell Harvester M-24R, Brandel Co., Gaithersburg, MD, USA) and two 5 ml washes with cold buffer through Whatman GF/C filter strips presoaked in 0.1% polyethyleneimine for at least three hours prior filtration, Specific binding was calculated as the difference in radioactivity

bound in presence and absence of either 1 yM substance P or 1 PM substance K

according to the 1 igand used, Binding of’the ligand to filters was quantitated

by counting filters in a LKB gamma counter with 80-85% efficiency, Under these conditions, specifically bound r lz51] substance P and 1 12511substance K

represent 86% and 64% of the bound ligand at half Kd values, respectively,

For autoradiography, guinea pig brain sections were prepared as described

before (30), Briefly, 500 g male guinea pigs were killed by decapitation and

their brains were rapidly isolated and immersed in 2-methyl butane (Kodak

Chemicals) at -40°C, mounted on cryostat chucks, and cut into 25 pm-thick

sections at -14OC. Sections were thaw-mounted near the edge of precleaned

gelatin-coated slides, air-dried on ice for 2 hours and then stored at -80OC

unti 1 used. Frozen slide-mounted brain sections were preincubated for 15 min in 50 r&l Tris.HCl and 0.2 mg/ml BSA, at pH 7.4 at 25'C and then incubated for 90 min as described above for membrane binding assays. At the end of the

192

incubation, slides were placed in racks and transferred sequentially through four rinses (1 min each) of 50 ml4 Tris.HCl, pH 7.4 at 4’C. Specific binding of either substance P or substance K was calculated as described above for membrane binding assays. Incubated slides were then rapidly dried under a stream of cold air and juxtaposed tightly against tri ti um-sensi tive fi lm (Ultrofi lm, LKB) and stored at room temperature for 7- 15 days, After this exposure, films were developed and processed as described before (30) D All peptides were obtained from Peninsula Labs, Palto Alto, CA.

RESULTS

Table 1 summarizes the data on substance P and substance K binding in guinea pig brain. The affinity (Kd) of substance P (2.1 nM) is greater than that of substance Kd (10.6 nM). On the other hand, substance K apparently labels a greater number of sites (Bmax) than substance P. This suggested to us that these two tachykinins might bind to different receptor sites in the guinea pig brain.

TABLE 1

Comparative affinity (Kd) and number of sites (Bmax) for [1251]Bolton-Hunter *substance P and [ ‘2511Bolton-Hunter substance K in guinea pig brain membrane preparations.

Peptide Kd (nM)

Substance P 2.10

Substance K 10.6

Bmax (fmol/mg protein

37.0

50.3

Values are mean of two experiments, each in triplicate using 10 concentrations of labelled peptides ranging from 0.1 to 40 nM. All data were analyzed by computerized curve fitting system,

This hypothesis is strongly supported by the differential ligand selec- tivity pattern obtained for the two ligands (Table 2) e While substance P and physalaemin are potent displacers of [ 2511Bolton-Hunter substance P binding, they are very weak in displacing I 12511Bolton-Hunter substance K. On the other hand, substance K, kassinin and neuromedin K are much more active on [ 12’11 Bol ton-Hunter substance K than [ 1251]Bol ton-Hunter substance P sites. Another tachykinin, eledoisin, appears to be fai rly non-selective for either site, Thus, the I igand selectivity pattern observed for [ ‘251]Bolton-Hunter substance P is substance P > physalaemin > eledoisin > substance K > kassinin > neuro- medin K while it is substance K > kassinin > neuromedin K > eledoisin > substance P > physalaemin for [ ‘251jBol ton-Hunter substance K,

193

TABLE 2

Comparative inhibitory potency of various tachykinins against [ 1251]Bolton- Hunter substance P and [ ‘251]Bolton-Hunter substance K binding in guinea pig brain membrane preparations

Tachyki ni n Substance P Substance K

Ki (nM) Relative Potency Ki (nM) Rel at i ve Potency

Substance P 1.4 100 1560 0.33 Substance K 1200 0,ll 100 Neuromedi n K 17,000 < 0,Ol 5x 1004 Kassinin 1515 0.08 2103 2404 Eledoisin 104 1.35 12.6 4,13 Physalaemin 407 25.8 3240 0,16

The values are the mean of three determinations, each in triplicate. The Ki values were calculated from the apparent Kd of both ligands using the formula Ki = ICso/l + F/Kd where ICso represents the concentration of peptides needed to displace 50% of specifically bound I ‘251]Bolton-Hunter substance P (0.8 nM) or [: 251]Bolton-Hunter substance K (3.5 nM), F is the concentration of free ligand in the incubation medium,

As shown in Figure 1 and 2, [ 1251]Bolton-Hunter substance P and [ 125~1 Bolton-Hunter substance K binding sites are heterogeneously and differentially distributed in guinea pig brain, Moderate to high densities of substance P sites are found in deep layers of the cortex (layers V and VI), caudate-putamen, central nucleus of the amygdala, habenula, ventral hippocampus, superior co1 1 i cul us, interpeduncular nucleus, locus coeruleus, parabrachial nucleus, trigeminal nucleus, inferior olive, nucleus tractus solitarius and dorsal horn of the spinal cord, Lower densities of sites are seen in the olfactory bulb, frontal cortex, septum, most hypothalamic nuclei, dorsal hippocampus, inferior colliculus and central gray matter, Very low densities of substance P binding sites are present in the thalamus,substantia nigra and cerebellum.

[ ‘251]substance K binding sites are highly concentrated in most cortical areas without apparent laminar distribution (Fig, 2). Other areas enriched in substance K binding sites are the caudate-putamen, septum, certain thalamic nuclei, hippocampus, superior colliculus, geniculate nucleus, substantia nigra and cerebellum. Moderate densities of sites are faund in theol factory bulb I central gray matter and dorsal horn of the spinal cord, Low to very low densities of sites are present in most hypothalamic and brain stem nuclei (Fig. 2). Only the nuc,leus tractus solitarius appears to contain moderate densities of substance K sites.

More specifically, the differential distribution of substance K and substance P binding sites is evident at the level of the hippocampus (Fig. 3). Substance P binding sites are more concentrated in the ventral portion of the hippocampus (Fig. 3b) while substance K sites appear more evenly distributed (Fig. 3a). Moreover, substance P binding is concentrated along the hippocampal fissure while substance K sites are distributed in a laminar fashion in the various layers of the hippocampus (Fig. 3a). At this

194

. . i

Figure 1. Photomicrographs of the autoradiographic distribution of [ 125~1 Bolton-Hunter substance P binding sites in guinea pig brain saggittal sect ions. Sections were incubated as described in text in the absence (A) or presence (6) of 1 uM substance P. cal losum;

Abbreviations: BS, brain stem; CC, corpus

colliculus; CE, cerebellum; CP, caudate-putamen; HI, hippocampus, IC, inferior

CB, olfactory bulb and SC, superior colliculus.

195

Figure 2. Photomicrographs of the autoradiographic distribution of [ 12511 Bolton-Hunter substance K binding sites in guinea pig brain saggittal sections. Sections were incubated as described in text in the absence (A) o presence (B) of 1 uM substance K. Abbreviations: CC, corpus callosum; CE, cerebellum; CP, caudate-putamen; FR, frontal cortex; Hi, hippocampus; OB, olfactory bulb; SC, superior colliculus; SN, substantia nigra and TH, tha I amus.

196

Figure 3. Photomicrographs of the autoradiographic distribution of [lz511) Bolton-Hunter substance K (A) and [ 1251]Bolton-Hunter substance P (B) binding sites in guinea pig brain coronal sections at the level of the hippocampus

(31). Sections were incubated as described in text. Abbreviations: AH, amygdalohyppocampal area; CC, corpus callosum; D, deep layers (layer V and VI) of the cortex; DLG, dorso-lateral geniculate nucleus and VH, ventral hippocampus.

level, high densities of substance K binding are also found in the dorsal and ventral lateral geniculate nucl.ei (Fig. 3a). Only very low densities of substance P binding are seen in these areas (Fig. 3b).

The differential distribution of substance P and substance K binding sites is also evident in the guinea pig substantia nigra (Fig. 4). While moderate to high densities of substance K binding are present in the sub- stantia nigra (Fig. 4a), substance P binding is virtually absent (Fig. 4b). The rat substantia nigra is also enriched in substance K binding sites (Fig. 4~).

197

Figure 4. Photomicrographs of the autoradiographic distribution of [ “‘11 Bolton-Hunter substance K (A) and [ 1251]Bo1ton-Hunter substance P (B) binding sites in guinea pig brain corona1 sections at the level of the substantia nigra (31). The distribution of [ 12511Bol ton-Hunter substance K binding sites at the level of the rat substantia nigra is also shown (C). Sections, were incubated as described in text., Abbreviations: CC, corpus cal losum; CG, central gray; HI, hippocampus; IP, interpeduncular nucleus; MG, medial geniculate nucleus; SC, superior colliculus and SN, substantia nigra.

Finally, high densities of substance K binding sites are found in the cerebellum (Fig. 5a) (lobules 9 and 10, especially) while only very low to low density of substance P binding sites is present in this structure (Fig. 5b). High densities of substance P binding sites are seen in the facial nucleus, parvocellular reticular nucleus and medial vestibular nucleus (Fig. 5b).

198

A

Figure 5. Photomicrographs of the autoradiographic distribution of [ 125~l Bolton-Hunter substance K (A) and [ 1251]Bolton-Hunter substance P (B) binding sites in guinea pig brain coronal sections at the level of the cerebellum. Sections were incubated as described in text. Abbreviations: IO, inferior olive; MVE, medial vestibular nucleus; PCRT, parvocel lular reticular nucleus; SOL, nucleus of the solitary tract; F, facial nucleus; 8, lobule 8 of the cerebellum and 10, lobule 10 of the cerebellum,

DISCUSSION

Our results clearly demonstrate that substance P and substance K bind to different populations of sites in the guinea pig brain. First, the relative affinity (Kd) and number of sites (Bmax) labelled by the two 1 igands are different. Substance P values are similar to those previously reported in rat brain using various ligands (15-19,22). Data on substance K binding characteristics (Kd and Bmax) have yet to be reported for other mammalian species. However, Beaujouan et al (20) have recently shown that [‘2511Bolton- -- Hunter eledoisin binds to an apparent single class of sites in rat brain synap- tosomes with an affinity similar to the one observed for substance K in guinea

199

pig brain. Further experiments should determine if [lz511 Bol ton-Hunter eledoisin binds to exactly the same population of sites as substance K in brain. It has already been suggested that these two ligands may label dif- ferent classes of tachykinin receptors, at least in peripheral tissues (32),

The differential ligand selectivity pattern observed for substance P and substance K also strongly suggests the existence of multiple tachykinin receptors in guinea pig brain. Substance P and physalaemin are active on substance P binding sites while they are very weak on substance K sites, On the other hand, kassinin, neuromedin K and substance K are potent on substance K sites but very weak on substance P binding sites. Similar results have been obtained in rat brain membrane preparations using various radiolabelled probes (12,15-22,27,33). Moreover, our data also demonstrate that neuro- medin K is fairly selective for substance K binding sites (15) while phy- salaemin is selective for substance P binding sites. Eledoisin is not selec- &ive for either type and may not be an appropriate ligand to characterize these two tachykinin receptor sub-types.

The relevance of the two 1 igand selectivity pattern observed in our experiments to the proposed substance P-P and substance P-E receptor classi- fication (9) remains to be determined. However, the poor selectivity of eledoisin toward either the substance P or substance K binding site suggests that this classification scheme might not be applicable to tissues such as the guinea pig brain. It would seem more appropriate to suggest the existence of at least two classes of tachykinin receptors in guinea pig brain, the subs- tance P and the substance K sites, respectively (12).

The differential autoradiographic distribution of substance P and subs- tance K binding sites also strongly support the existence of multiple classes of tachykinin receptors in guinea pig brain. The overall distribution of subs- tance P binding sites in this species is similar to the one reported in rat brain (22-26) with high densities of sites found in the striatum, habenula, amygdala, dendate gyrus, superior colliculus and locus coeruleus, and very low densities seen in the substantia nigra.

However , it appears that substance K binding sites could be more extens- ively distributed in guinea pig than in rat brain. In rat brain, substance K binding sites are highly concentrated in very few areas such as layer IV of the cerebral cortex, substantia nigra, few hypothalamic nuclei and nucleus tractus sol i tari us (28) o On the other hand, substance K binding sites are highly concentrated in various guinea pig brain regions, such as the cortex, striatum, certain thalamic nucleus, hippocampus, superior colliculus, subs- tantia nigra and cerebellum. This suggests that substance K may have a more extensive role on the control of brain functions in guinea pig.

The high densities of substance K binding sites present in the substantia nigra is of special interest, Previously, we (12,22-24) and others (2.5,26), have reported on the absence of substance P binding sites in the rat subs- tantia nigra. This is true of the guinea pig brain as well, and is very sur- prising since high levels of substance P-like immunoreactivity are present in the substantia nigra. The immunoreactive substance P is located in terminals originating from cell bodies located in the striatum (24,341, Thus, the nigral substance P released from these terminals would be expected to exert various

200

effects in this structure. However very negligible electrophysiological

effects of substance P on substantia nigra pars compacta dopaminergic cell

bodies have been reported (35). Since substance P and substance K are

similarly distributed in brain and are derived from the same precursor (6),

we suggest that the most active tachykinin in the substantia nigra could be

substance K. This hypothesis is supported by the high density of substance

K binding sites present in the substantia nigra and by recent data showing

that substance K possesses potent electrophysiological (36) and biochemical

(12,37) effects in this structure. Thus, further experiments on the relative

importance of these two tachykinins in the striatonigral pathway are urgently

needed.

The presence of high densities of substance K binding sites in the cere-

bellum was unexpected. To our knowledge, only very low levels of tachykinin-

I ike immunoreactivi ty have been found in the cerebel lum (24,38-40) except in

neonatal rats (41). Thus, possible species of difference should be considered

and the existence of substance K-like immunoreactivity in the guinea pig cere-

bel lum should be investigated.

In summary, [ ‘2511Bolton-Hunter substance P and [1251]Bolton-Hunter subs-

tance K label two different classes of sites in guinea pig brain: a substance

P site and a substance K site. Moreover, these two sub-types of tachykinin

receptors are differentially distributed in guinea pig brain, Finally, the

high density of substance K binding sites in the substantia nigra suggests

that this peptide could be the most active tachykinin in the striatonigral

pathway.

ACKNOWLEDGEMENT

This research was supported by an Establishement Grant from the “Fends

de la Recherche en Sante du Quebec (FRSQ)“. Dr. RCmi Quirion is a “Chercheur-

Boursier” from the FRSQ. We wish to thank Mrs. Joan Currie for her expert

secretarial assistance.

I.

2,

3.

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and ways of entry to the cerebellum, Neuroscience, 7, 639-645.

Accepted 21/z/85

204