Neuropeptides 4: 325-329, 1984

COMPARATIVE POTENCIES OF SUBSTANCE P, SUBS SUBSTANCE P RECEPTORS.

TANCE K AND NEUROMEDIN K ON BRAIN

Remi Qui rion and Carmenci ta Pi lapi 1. Doug las Hospital Research Centre,

6875 Blvd. LaSal le. Verdun, Quebec, Canada H4H lR3.

ABSTRACT

The relative potency of substance P, substance K and neuromedin K on C3Hlsubstance P binding has been compared in rat brain membrane prepara- tions. Substance K and neuromedin K, two newly isolated substance P- related mammal ian peptides, are very weak competitors for E3Hlsubstance P binding sites. It suggests that these two neuropeptides are not acting on the same receptor type as substance P. Moreover, previous substance P receptor classifications are unlikely to be applicable to brain tachykinin receptor sub-types.

INTRODUCTION

Besides substance P, recent reports have demonstrated the existence of other tachykinins in mammalian brain (I-3). It has been shown that two peptides related to the tachykinin, kassinin, are present in the mammalian spinal cord. The first one, substance K or a-neurokinin has been isolated from both porcine and bovine spinal cord. The other one, neuromedi n K (B-neurokinin) has only been isolated from porcine spinal cord, thus far. Moreover, Nawa et al (4) have recently reported the nucleotide sequence of -- two substance P precursors present in bovine brain. A precursor contains one copy each of substance P and substance K whi le the other wi 11 most likely generate only substance P. A putative prohormone for neuromedin K has yet to be characterized.

On the basis of results obtained in various bioassays, it has been suggested that substance P interacts with more than one class of receptors (S-6) c Lee et al (7) have suggested the existence of substance P-P (for physalaemin)

-- and substance P-E (for eledoisin) receptor sub-types. On the

fi rst sub-type, substance P, physalaemin, eledoisin and kassinin are more or less equipotent while kassinin and eledoisin are much more active than substance P itself on the substance P-E receptor. Using E3HJsubstance P as the 1 igand, we have reported the characterization of brain binding sites that are related but not identical to substance P-P receptors (8). We now report that substance K and neuromedin K are weak competitors for brain substance P receptor label led with C3Hlsubstance P. The relevance of these findings for brain substance P receptor classification is discussed.

325

MATERIALS AND METHODS

Whole brains (minus cerebelli) were removed on ice from 200 g male Sprague-Dawley rats (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 KC1 using a Brickman polytron (setting 6, 20 set). The homogenate was then centrifuged for 10 min at 50,000 g. The supernatant was dis&arded and the pellet resuspended in 50 mM Tris.HCl buffer, pH 7.4 at 4 C plus 300 mM KC1 and 10 mM EDTA.Kp before incubation on ice for 30 min with gentle agitation. After centrifugation as above, the pellet was resuspended in 20 volumes of 50 m!l 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.

For binding assays 200 ul of membrane suspension were incubated for 20 min at 20°C with 200 ~1 of a 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), 1.00 nM of C3Hlsubstance P (23.8 Ci/mmole, New England Nuclear) and various substance P homologues as indicated, Incubations were term- inated by rapid filtration (Cell Harvester M-24R, Brandel Co., Gaithersburg, USA) and two 5 ml washes with cold buffer through Whatman GF/C filter- strips presoaked in 0.1% polyethyleneimine for at least 3 hours prior to use. Specific binding was calculated as the difference in radioactivity bound in presence and absence of 1 )JM substance P. Binding of the triti- ated ligand to filters was quantitated by counting filters in 6 ml Scinti- Verse I I (Fisher Scientific Ltd) scintillation cocktail. Under these conditions, specifically bound C3HIsubstance P represents between 75-85% of totally bound C’HIkubstance P. All peptides were obtained from Peninsula Labs, Belmont, CA.

RESULTS AND DISCUSSION

As shown in Table 1, substance P itself is the most potent competitor for sites labelled by [3Hlsubstance P in rat brain. Physalaemin also possess good affinity for these sites while eledoisin and kassinin are respectively 300 and 1500 times less potent than substance P in inter- acting with [3Hlsubstance P binding. These results are similar to those reported before in brain sections (8-11) and membrane preparations (12-15).

The two newly isolated tachykinins, substance K and neuromedin K appear to differentially interact with these binding sites. Substance K is a weak competitor against C3Hlsubstance P binding while neuromedin K is almost totally inactive (Table 1). It suggests that substance K and neuromedin K are not endogenous ligands for this central substance P receptor sub-type. Hill coefficients are close to unity for all homologues except substance K which may suggest complex interactions between this peptide and C3H1 substance P binding sites in the central nervous system (Table 1).

326

TABLE

I R

elat

ive

affi

nit

ies

of

vari

ou

s su

bst

ance

P

h

om

olo

gu

es

for

[3H

]

sub

stan

ce

P

bin

din

g

site

s p

rese

nt

in

rat

bra

in

mem

bra

nes

Pep

t i

de

Pri

mar

y S

tru

ctu

re

Ki

(nM

) lli

1

I C

oef

fici

ent

Rel

ativ

e P

ote

ncy

Su

bst

ance

P

A

rg-P

ro-L

ys-P

ro-G

ln-G

ln-P

he-

Ph

e-G

ly-L

eu-M

et-N

H?

--

-

Su

bst

ance

K

(a

-Neu

roki

nin

) H

is-L

ys-J

hr-

Asp

-Ser

-Ph

e-V

al-G

ly-L

eu-M

et-N

H2

--

Neu

rom

edi

n

K

(B-N

euro

kin

in)

Asp

-Met

-His

-Asp

-Ph

e-P

he-

Val

-Gly

-Leu

-Met

-NH

2 --

-

Kas

sin

in

Asp

-Val

-Pro

-Lys

-Ser

-Asp

-Gln

-Ph

e-V

al-G

ly-L

eu-M

et-N

H~

--

Ele

do

isin

p

Gl

u-P

ro-S

er-L

ys-A

sp-A

la-&

-l

le-Q

-Leu

-Met

-NH

2 --

Ph

ysal

aem

in

pG

lu-A

la-A

sp-P

ro-A

sn-

Lys

-Ph

e-T

yr-C

ly-L

eu-M

et-N

H2

--

0.25

f 0.03

0.86

+ 0.05

100

540

f 76.2

0.69

t 0.04

0.05

>2000

<O.Ol

365

f. 26-3

0.79

f 0.06

0.07

86.0 *

11.6

0.81

t 0.04

0.30

0.63

+ 0

04

0.92

f 0-06

39.7

Th

e va

lues

ar

e th

e m

ean

+

S

.E.M

. o

f th

ree

det

erm

inat

ion

s,

each

in

tr

iplic

ate.

Th

e K

i va

lues

w

ere

calc

ula

ted

fr

om

th

e ap

par

ent

Kd

(0

.37

nM

) o

f C

3HIs

ub

stan

ce

P

usi

ng

th

e fo

rmu

la

Ki

=

ICss

/l +

F

/Kd

lcsO

re

pre

sen

ts

the

con

cen

trat

ion

o

f p

epti

des

n

eed

ed

to

dis

pla

ce

50%

o

f sp

ecif

ical

ly

bo

un

d

C3H

lsu

bst

ance

P

an

d

F

is

the

con

cen

trat

ion

o

f fr

ee

ligan

d

in

the

incu

bat

ion

.

As mentioned above, C’HJsubstance P most likely labels a substance

P-P-“1 i ke” receptor sub-type in the brain (8). This could explain the

relatively poor affinity of substance K for C3Hlsubstance P binding sites-

However, some important differences exist between central and peripheral

receptor sub-types. For example, substance P, eledoisin and kassinin

are almost equipotent on substance P-P receptors in peripheral tissues

(7). The last two homologues are much weaker than substance P on the

brain receptor sub-type (8-15). Thus,eledoisin, kassinin, substance K

(16,17) and neuromedin K appear to be much mOre active on peripheral

than central substance P receptors. These data show that the “peripheral”

substance P-P and substance P-E receptor classification (7) does not

appear to be fully applicable to brain substance P receptors, The cha rac-

terization of a putative brain “tachykinin-kassinin like” receptor using

radiolabel led substance K should reveal if these sites are related or not

to the substance P-E receptor sub-type. It also suggests that substance

P itself is the only endogenous ligand known thus far for the mammalian

brain C3Hlsubstance P receptor

In su

neuromedi n

in rat bra

endogenous

investigat

mav, the two newly isolated tachykinins, substance K and

K show very poor affinity for C3HJsubstance P binding sites

in. Thus, these two neuropept i des are more I ikely to act as

ligands of a kassinin-related brain receptor. We are currently

ing this exciting possibility,

ACKNOWLEDGEMENT

This research was supported by the Douglas Hospital Research Centre.

Remi Quirion is a scholar of the “Fends de la Recherche en Sante du QuCbec”.

We wish to thank Mrs. Joan Currie for expert secretarial assistance.

1,

2 I,

3.

4.

6.

REFERENCES

Kimura, S., Okada, M,, Sugita, Y., Kanazawa, I. and Munekata, E. (1983).

Novel neuropeptides, neurokinin CY and 3, isolated from porcine spinal

cord, Proc. Jap. Acad, Ser. 9. 59:101-104.

Maggio, J.E., Sandberg, B.E.B., Bradley, C.V., Iversen, L.L., Santikarn,

S ., Williams, B.H., Hunter, J.C. and Hanley, M.R. (1983)~ Substance K:

a novel tachykinin in mammalian spinal cord. In: Substance P- Dublin

1983. P. Skrabanek and D. Powell (Eds) , Boole Press, 20-21.

Kangawa, K., Minamino, N., Fukuda, A. and Matsuo, H. (1983); Neuro-

medin K: a novel mammalian tachykinin identified in porcine spinal

cord, Biochem. Biophys. Res. Commun. 114:533-540.

Nawa, H., Hi rose, T., Takashima, H., Inayama, S,. and Nakanishi, S., (1983). Nucleotide sequences of cloned cDNAs for two types of bovine

brain substance P precursor, Nature 206:32-36.

Sandberg, B_E.B. and Iversen, L.L. (1982). Substance P. J. Med. Chem.

25:1009-1015.

Couture, R, and Regal i, D, (1982). Smooth muscle pharmacology of subs-

tance P, Pharmacology, 24: l-25-

328

7.

8.

9.

IO.

11.

12.

13.

14. Cascieri, M-A, and Liang; T, (1983). Characterization of the substance P receptor in rat brain cortex membranes and the inhibition of radio-

ligand binding by guanine nucleotides. J. Biol. Chem.. 258:5158-5164,

15. Park, C,H., Massari, J., Quirion, R,., Tizabi, Y., Shults, C,W., Chase,

T. and O’Donohue, T,L, (1984). Characterization of 3H-substance P

binding sites in rat brain membranes. Peptides, in press,

16, Hunter, J.C. and Maggio, J-E. (1984)” Pharmacological characterization

of a novel tachykinin isolated from mammalian spinal cord,, Eur. J. Pharmacol. 97:159-160.

17. Nawa, H., Doteuchi, M., tgano, K., Inouye, K. and Nakanishi, S. 0984)..

Substance K: a novel mammalian tachykinin that differs from substance P

in its pharmacological profile, Life Sci., in press.

Lee, C.M,, lversen, L,L,, tianley, M.R, and Sandberg, B.E.B, (1982).

The possible existence of multiple receptors for substance P. Naunyn-

Schmiedeb. Arch, Pharmacol i 318:281-287,

Quirion, R,, Shults, C.W., Moody, T-W., Pert, C.B., Chase, T.B. and

O’Donohue, T,L, (1983) c Autoradiographic distribution of substance P

receptors in rat central nervous system, Nature 303:714-716.

Qui rion, R., Shults, C.W, , Moody, T,W,, Wolf, S.S., Jensen, R.T.,

Pert, C.B., Chase, T.B. and O’Donohue, T.L. (1983). Autoradiographic

localization of substance P (SP) receptors in rat brain: a comparison

of binding properties of L3H7SP, C’2511SP and ~‘2511physalaemin~~ In:

substance P- Dublin 1983. P. Skrabanek and D. Powell (Eds), Boole

Press, 55-56.

Shults, C.W., Qui rion, R,, Chronwal I, B., Chase, T.B. and O’Donohue,

T-L. (1984). A comparison of substance P and substance P receptors

in the rat central nervous system. Peptides, in press.

Wolf, S.S., Moody, T.W., Quirion, R. and O’Donohue, T.L, (1984). Bio-

chemical characterization and autoradiographic localization of central

substance P receptors using ‘251-physalaemin, Brain Res. submitted,

Viger, A., Beaujouan, J.C., Torrens, Y. and Glowinski, J, (1983).

Specific binding of a 1251-substance P derivative to rat brain

synoptosomes. J. Neurochem. 40: 1030-1039.

Torrens, Y., Beau’ouan, J.C,, Viger, A. and Glowinski, J. (1983).

Properties of a li5 I-substance P derivative binding to synaptosomes

from various brain structures and the spinal cord of the rat. Naunyn-

Schmiedeberg’s Arch. Pharmacol, 324:134-139.

Accepted 17/5/84

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