Neuromedin U-8 and U-25: Novel uterus stimulating and hypertensive peptides identified in porcine spinal cord

Vol. 130, No. 3, 1985 August

BIOCHEMICAL

15, 1985

NEUROMEDIN U-8

AND U-25: Naoto

Received

June

27,

NOVEL UTERUS STIMULATING AND HYPERTENSIVE PEPTIDES IDENTIFIED IN PORCINE SPINAL CORD

MINAMINO,

Department

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1078-l 085

Kenji

KANGAWA and Hisayuki

MATSUO

of Biochemistry, Miyazaki Medical Kiyotake, Miyazaki 889-16, Japan

College,

1985

SUMMARY: Two novel peptides eliciting a potent stimulant effect on the rat uterus smooth muscle have been purified and identified in porcine spinal cord. These peptides were designated as neuromedin u-8 (8 amino acids long) and U-25 (25 amino acids long) refering to their uterus stimulating activity. Sequence analyses and syntheses revealed that neuromedin U-8 is a novel octapeptide with a C-terminal amide structure, while u-25 contains the U-8 sequence at its C-terminus, preceded by paired Arg residues, implicating their biosynthetic relationship. Their potent uterus stimulating activity and hypertensive effect, as well as their unique C-terminal amide structure are indicative of their specialized physiological function. @ 1985 Academic Press, Inc.

In a survey a

simple

preparations muscle

but

for

unidentified

sensitive

assay

successfully

stimulating "B" ,

enabled

peptides

comprising

and

the complete

amino

acids

uterus

activity,

Their

purified. be discussed

us

spinal "KM

acid

sequence

and 11U-25" by

hypertensive

in comparison

we present

long)

which those

effect

that

on smooth-muscle

a family

of novel

smooth-

cord, named as "neuromedins", "L" ckassinin-like) and "Nl!

the

identif

ication

in porcine

of two new neuromedins.

spinal

designated

(25 amino

acids

long)

peptides

have

been monitored

both

and smooth-muscle with

we have demonstrated

stimulant

to discover

in porcine Here

as IIU-!j'lt (8 amino stimulant

the

"C"(bombesin-like)?

(neurotensin-like)(l-5). cord

neuropeptides, for

of several

stimulant

activities

known bioactive

after will

their and also

peptides.

MATERIALS AND METHODS in the present purification were Purification: The starting materials used side fractions obtained in our previous purification of neuromedin B, C, K, L Porcine spinal cords (ca. 20kg obtained from 550 pigs) were and N (l-5). minced, heat-treated and extracted with 1M CH COOH containing 20mM HCl. After by acetone-precipitation an Amicon UH-2 membrane, aallowed desalting through was evaporated at a final concentration of 752, the supernatant --in vacua to

Abbreviations: TLC, thin layer substance. P; polypeptide: Mr,

RP-HPLC, reverse phase high performance liquid chromatography: TFA, trifluoroacetic acid; BK, bradykinin; SP, chromatography; vasoactive intestinal polypeptide: PP, pancreatic VIP, molecular weight.

0006-291X/85$1.50 Copyright AN rights

0 1985 by Academic Press, Inc. of reproduction in any form reserved.

1078

Vol.

130,

BIOCHEMICAL

No. 3, 1985

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

materials were dissolved in 1M CH COOH and then submitted to dryness. Dry ? m), pre-equilibrated with batchwise chromatography on SP-Sephadex C-25(H+-fo 1M CH COOH. Successive elutions with 1M CH COOK, 2M pyridine and 2M pyridineCH COdH (pH 5.0) yielded three fractions of’SP-I, SP-II and SP-III. The SP-III frdction containing basic peptides was separated by gel filtration on a column of Sephadex G-50 and then on a column of Sephadex G-25. An aliquot of each Uterus fraction was subjected to bioassay for rat uterus stimulant activity. activity was observed in a wide range of fractions A to F (Fig. la). Neuromedin 11-x and U-25 were isolated from fraction F and fraction 8, respectively. i) Neuromedin U-8: After lyophilization, fraction F was subjected to ion exchange HPLC (Fig. lb). The uterus active fraction eluted at 103-105 min was further purified by RP-HPLC to vield neuromedin U-3 in a pure state (Fig. lcl. ii) Nenromedin U-25: Fraction-B (Fig. la) was submitted to a preparative RP-HPLC to yield two peaks with uterus activity (Fig. 2a). The uterus active fraction eluted at 128-136 min was further purified by cation exchange HPLC to give a single bioactive peak eluted at 68-71 min (Fig. 2b). The active fraction obtained above was further separated by RP-HPLC on a Chemcosorb 7-diphenyl column (Chemco) and finally on a C-13 column to purify neuromedin U-25 to homogeneitv(Fig. 2~). Structure Analyses: i) Amino acid composition: Analyses were carried out with an amino acid analvzer (Hitachi-835), after acid hvdrolvsis of the purified neuromedins in 6M HCl containing 0.17:’ phenol and 0 .G27! 2-mercaptoethanol at 110°C for 20 hr. ii) Amino acid sequence: Neuromedin U-8 (ca. 200 pmol) and neuromedin U-25 (ca. 200 pmoll were each sequenced with a gas-phase sequencer (Applied Biosystems Model 47DAl (6), coupled with HPLC identification of the resulting PTH-amino acids (2). iii) C-Terminal analysis: The C-terminal amide structure W&S verified by trypsinization of the pept ide , followed by dansplat ion, in a manner similar to the method reported by Tatemoto and Mutt

(7). Syntheses : Neuromedin U-8 and U-25 were synthesized by solid phase techniques conducted on p-methyl-benzhydrylamine resin. Bioassay: i) Uterus stimulant activity: Assay was performed according to the described method (8) by using strips of rat uterus preparation freshly isolated in proestrous stage, in a modified Locke’s solution at 32°C. ii) Hypertensive effect: Systemic blood pressure was measured from the carotid artery in rats (000-4@Og) anesthetized with pentobarbital (5). Indicated amounts of peptides in 100-200 ul of 0.4’7 saline were administered through a cannula in the femoral vein. BK, SP and VIP were purchased from Protein Research Foundation. Bovine PP was kindly donated from Dr. A. Arimura, Tulane University School of Medicine.

RESULTS Neuromedin for

the

manner

to

peptide

that

used

Sephadex

of

G-50

further

uterus

to

collect

the

regions

(A-F). three

spinal fractions

purified subjected

was

(1).

The the

rat

spinal uterus

by

SP-Sephadex was

a Sephadex widely

(Fig.

of

C-25

to G-25

bioactive subsequent

Yr

1079

As in

peak

of

major

to

The

ca.

basicity purify

basic of

in

the on

daltons, shown

the

peak,

higher

assay

gel-filtration

5,000

F (tir the

RP-HPLC

(I-5).

by

column.

fraction

an

a similar

chromatography

separated

From

using in

neuromedins

distributed

lb).

cord

preparations

of

cord

HPLC

peaks

to

porcine

corresponding on

Cation-exchange bioactive

from

isolations

prepared

activity

DISCUSSION

isolate?

previous

rechromatographed

yielded

was

the

porcine

stimulant

previously

in

on

(SP-III!,

extract

were

purified

effect

fraction

acid

min

Us were

stimulant

AND

which

Fig.

la,

chromatographic 1,000

daltons)

neuromedin eluted neuromedin

B was at U-8

104 to

Vol.

130,

No. 3, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

Abd---- B

C

DE

RESEARCH

COMMUNICATIONS

F&

L

0

10

40 number

30

20

Fraction

Time

50

60

70

(mtn)

C

-.

0

_- - _____

10

____-__-- ---

20

________--------

30

Tlme

_ __------ _______-------

40

‘i z II 0:

50

(mln)

Fig. 1 Purification of neuromedin u-8. a) filtration of SP-III on a Sephadex G-25(fine) column (7.5 x 135 cm). Eluent: 1M CH COOH. Fraction size: 100 ml/tube. Flow rate: 60 ml/hr b) Cation exe a ange HPLC of fraction F (l/3 portion) in Fig. la. Column: TSK IEX-530 CM (4.0 x 300 mm, Toyosoda). Flow rate: 1 ml/min. Solvent system: Linear gradient elution from A:B = 100: 0 to 85:15 for 48 min, followed by the second gradient from A:B = 85:15 to 50:50 for 48 min. (A) 1OmM HCOONHd(pH 6.5):CH3CN ; 9O:lO (v/v), (B) l.OM HCOONH4(pH 6.5):CH3CN = 9O:lO (v/v,. 1080

Vol.

130,

No. 3. 1985

homogeneity

BIOCHEMICAL

(Fig.

fraction

B in

Fig.

fraction

B on

a C-18

(Note

that

from

purified.) min

peak

2~).

II-6

and

using

1.05

a

for

U-5

and

3b).

in

The

were

verified

the

structures

native

proceeded

by

acid

of

showr (0)

comparison.

structure

been

has

PP.

(7).

*Neuromedin U-8: Asp 1.05(l), Pro Neuromedin (J-25: .Asp .1.07( n,, Ser Ile i?.30(1), Leu

the

the

related far

peptides observed As oc

C-terminal

exhibiting in

seen

I:ig.

uring

in

Leu

0.04

O.o711), 1.00(l),

Glu Tyr

3.89(4J, 0.92(11,

1).

the

peptides,

end

(Fig.

U-8

and

,ja U-25 of

the

of

complete

are

U-3

the

shown or

C-terminal

hormonal

the

pancreatic

(10)

neuromedin

or

releasing

and

U-25 amide

physiological factors

a partial II-‘,

of

and

C-terminal U-25 to

also

is

O.oOil),

Phe

2.0%?(2),

Arg

2.02(?).

Pro

2.27(Z),

Phe

4.04(41,

Gly Lys

1.00(l), l.O5il),

Val Arg

1.74(2), 4.14(4)

Tyr

known

is

structure

c) Final purification of neuromedin U-8 by RP-HPLC. Column: Chemcosorb SODS-H (4.0 x 250 mm, Chemco). Flow rate: 1 ml/min. Solvent: Linear gradient elution from (A) to (B) for 80 min. Hz0 : CH3CN : 10% TFA = (A) 90 : 10 : 1, (8) 40 : 60 : 1 (v/v). uterus activity (Fig.la,b) and uterus active fraction (Fig.lc) are shown by black bars. BSA, bovine serum albumin; SPO, SP sulfoxide; gNE, cyneo-endorphin; SS, snmatostatin; LE, Leu-enkephalin; R6LE, [Ax-g I-Leuenkephalin; LHRH, luteinizing hormone releasing hormone; Dyn8, dynorphin[l-81. 1081

acid Edman

unambiguously

sequences

neuromedin

of

confirmation

been

3c .

for

sequences

PTH-amino

Thus,

to

acid

nmol

0.84

comparison

hypothalamic in

column

analysis.

neuromedin

have

by

amino

carboxyl

identified.

asparaginamide

1.00(l),

of

polypeptidc(VIPI

highly so

25

Positive

known

C-15

the

neuromedin

the

ones.

intestinal

of been

-Arg-Pro-Arg-X-CONH, But )

where

each

of

of

(J-25

a

acid

chromatographic

and

state

as

of

the

(71.

synthetic C-8

peptides

feature often

hormones of

the

pmol step

Mutt by

corresponding

.Tc .

pm01

upto

a pure

and

Amino

structure and

provided

vasoactive

150

3

128-1.16 From

on

estimated

first

amide

sequences in

a unique

gastrointestinal

in

and

200

yields

Tatemoto

Fig.

with

the

repetitive

was

pigs.

About at

neuromedin in

found

as

sequence

sequencer.

also

200

in

at

was

2b).

footnote)*,

comprise

Za).

peptide

finally (see

of

(Fig.

eluted

isolated

and

were from

determined

of

(Fig.

from

RP-HPLC

releasing peak

to

isolated

peaks

activity was

was

daltons.

bioactive

data

yields

starting

Although

is

their

above their

polppeptide(PP)

activities,

found

asparagine

with

as

never

were

high

sequences

established,

above

method

deduced

peptides

amino

the

hioactive

column,

peptide

C-terminal

two

U-25

analyses

automated each

gave

first

acid

were

from

the

amino

U-25, U-25

a gas-phase

of

diphenyl

the

3,000

gastrin

neuromedin

and

nmol

degradation

found

on

ca.

later,

COMMUNICATIONS

U-25

Mr

stimulating

above,

purified

generated

have

HPLC

respectively,

and

neuromedin

for

uterus

on

II-25

residues,

and

of

obtained

Based

to

eluted

RESEARCH

neuromedin

column, peak

peak

BIOPHYSICAL

hand,

corresponding

second

RP-HPLC

(Fig.

other

preparative

the

a single

successive

was

la,

Cation-exchange

gave

binactive

U-8

On the

ICI.

AND

occur

Vol. 130, No. 3, 1985

8lOCHEMlCAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Tlme

b

2 1 J

5 J

g 0.05

H 5 J

(min)

L JJ

a% JJ

___----

_----

-

8 p‘1

--___----

ii x i %

'

O;-; 0

20

40

60 (mln)

Tlme

0.15

80

100

c

I

P 0GO,lO_---

______--

----

________--

------

_____----

______- -----

_______--

-----

E 0" g 0.05 % I O-

I 10

60 20

30 Time

(mln)

40

50

Fig. 2. Purification of neuromedin U-25. a) Preparative RP-HPLC of fraction B (l/7 portion) in Fig. la. c01uml: TSK ODS-120A (2.0 x 25 cm, Toyosoda). Flow rate: 5 ml/min. Solvent system: Linear gradient elution from A:B =90:10 to A:B = 30:70 for 250 min. Solvents A and B were as in Fig. lc. b) Cation exchange HPLC of uterus active fraction (l/2 portion) cluted at 128-136 min in Fig. 2a. Column: TSK CM-2SW (4.6 x 250 mm, Toyosada). Flow rate: 1 ml/min. Solvent system: Linear gradient elution from A:8 = 100:0 to A:B 2 SO:50 for 100 min. Solvents A and B were as in Fig. lb.

1082

Vol.

130.

BIOCHEMICAL

No. 3, 1985

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

b

10

5

15

20

25

Cycle

number

8

4

J

Tyr-Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH2

: NMU-8

Phe-Lys-Val-Asp-Glu-Glu-Phe-Gln-Giy-Pm-Ile-Val-Ser-G1n-Asn-Arg-Arg-Tyr-Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH~ -

: NMU-25

----Ala-Ala-Glu-Leu-Arg-Arg-Tyr-Ile-Asn-Met-Leu-Thr-Arg-Pro-Arg-Tyr-NH~ ---_--

: PP

----Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-~* -----_

: VIP

-

Fig. 3. Amino acid sequences of neuromedin U-8 and neuromedin a) Yield of PTH-amino acid generated from neuromedin u-8. b) Yield of PTH-amino acid generated from neuromedin U-25. c) Complete amino acid sequences of neuromedin U-8(NMU-8) compared with porcine PP and VIP. Homologous sequences are line indicates the paired basic signal for processing.

only

in

VIP.

The

and

neuromedin

U-25

processing

signal

most may

likely take

residues

in

generate

rat

systemic

blood

substance

P(SP)

(Fig.

basis), U-25

neuromedin is

repeated Furthermore,

three

times

twitching U-2j

of

as

were

4).

With

is

approximately

potent

of

uterus

was

found

to

at

so

with

far

of

rat

sensitivity

-la),

which

paired

basic as

of

contrcation

bradykinin(BK)

and

activity BK.

while

neuromcdin 11-25

while of

(molar

(J-8 rat

induced did

uterus

c) Final purification of neuromedin U-25 by RP-HPLC. Column : Chemcosorb 30DS-H (4.6 x 75 mm, Chemco). Flow rate: 1 ml/min. Temperature: Solvent system: Linear gradient elution from A:B = 8@:20 45°C. to A:B = 0:lOO for 192 min. Solvents A and B were as in Fig. ic. Relative uterus contractile activity (Fig. 2a,b) and uterus active fraction (Fig. 2~) were shown by black bars. 1083

is

amides

uterus

neuromedin (Fig.

typical

procrssinc

peptide

those

of

identified.

stimulating with

a U-6

site

been

Interestingly,

increase

is

a similar

the

Us on

uterus

C-terminus

neuromedin that

equipotent BK.

the

C-terminal

not

compared

preparation to

PP

neuromedin

regard

as

of

truncated have

at

which

suggests

and

they

pressure

u-8

occurence

respective

activities

located

and U-25(NMU-25) underlined. Dashed

structure.

[I-25

family

though

is

Ax-g-Arg

natural

VIP/PHI

even

biological

a

neuromedin

their

entities, The

The from

also

sequence

by

(11,12).

place to

C-8

preceded

processed

endogenous

and

neuromedin

U-25.

not. to

Vol.

130,

No. 3, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

a BK 1.0

-

NMU-8 1.0 nM

nM

\-

. 0

NMU-25

NMU-25 0.7 ntl

0.35

nM

0

2

4

and

11-25

. 2

4

.

- 0

2

4 Time

0

2

4

rat

uterus

imln)

b

Time

Fig.

4.

-

Biological effects blood pressure.

systemic

neuromedin

IIs.

essential part

for

These

of neuromedin

activity. fact

they

Furthermore, anesthetized

rats

hypotensive

effect. in

systolic

(J-25 was longer It

should

amide rat

be mentioned

C-terminus

at the or on

is

important

neuromedin

a concentration

blood

900

elicit

stimulation.

of

that

exerting

nM, t;ith

times

no appreciable

indicating that its biological

a contractile which

hut

effect

(I-8,

SP and HK show potent 1084

blood

short-lived

LI-5 (Fig.

4b).

lacking

an c1-

biological

on guinea

to

15 mmHg of neuromedin

than

the amide activities.

Ils.

about

effect

more potent

the

nmol/kg) arterial

induced

des-amid+neuromedin

, showed

pressure,

v-8

of the despite

in

strong

The hypertensive

three

the N-terminal

(1.0

increase

nmol/kg,

may he

neuromedin

(1-q

have

rat

prolongation

with

neuromedin which

of 3.0

synthetic

C-terminus for

uterine

and sustained

pressure.

U elicited of

not

RK and SF,

and about

and that and

similarity

and

U-h sequence

reinforcement

structural

a dose

blood

lasting

structure

uterus

neither

At

uterus.

for

a rapid

from

on

the neuromedin

on rat

injection

produced

differing

increase

some

u-8

that

effect

PP and VTP did have

intravenous

pressure,

indicate

stimulant

11-25 may serve

Note that that

of neuromedin

results

exerting

(min)

effects

on

structure at the Tncidentally, pig

ileum

stimulant

even at activity

BIOCHEMICAL

Vol. 130, No. 3, 1985

(0.4-1.0

nM).

The pharmacological

of SP and BK, function

--in vivo

strongly

suggest

as neuropeptides

AND BIOPHYSICAL

spectra that

of neuromedin

neuromedin

RESEARCH COMMUNICATIONS

Us distinct their

Us may have

from those specialized

or hormones.

This work was supported in part by a Grant-in-Aid from the ACKNOWLEDGEMENTS: Science and Culture of Japan. We sincerely thank Ministry of Education, Tamayo Hatoh and Masuyo Hiranaga of our Department for their Tetsuji Sudoh, technical assistance. REFERENCES 1. Minamino, N., Kangawa, K. & Matsuo, H. Biochem. Biophys. Res. Commun. 114, 541-548 (1983). 2. Gamine, N., Kangawa, K. & Matsuo, H. Biochem. Biophys. Res. Commun. 9, 14-20 (1984). 3. Kangawa, K., Minamino, N., Fukuda, A. & Matsuo, H. Biochem. Biophys. Res. Commun. 114, 533-540 (1983). 4. Minamino, N., Kangawa, K., Fukada, A. & Matsuo, H. Neuropeptides 4, 157-166 (1984). 5. Minamino, N., Kangawa, K. & Matsuo, H. Biochem. Biophys. Res. Commun. 122, 542-549 (1984). 6. Hewick, R.M., Hunkapiller, M.W., Hood, L.E. & Dreyer, W.J. J. Biol. Chem. 256, 7990-7997 (1981). (1978). 7. Tatemoto,K. & Mutt,V. Proc. Natl. Acad. Sci. U.S.A. 75, 4115-4119 8. Carraway, R. & Leeman, S. J. Biol. Chem. 248, 6854-68iil (1973). 9. Floyd, J.C. Jr., Fajans, S.S., Pek, S. & Chance, R.E. Recent Prog. Horm. Res. 33, 519-570 (1977). 10. Bodanszky, M., Klausner, Y.S. & Said, S.I. Proc. Natl. Acad. Sci. U.S.A. 70, 383-384 (1973). 11. Nakanishi, S. et al. Nature 3, 423-427 (1979). 12, Steiner, D.F. Quinn, P.S., Chan, S.J., Marsh, J. &. Tager, H.S. Ann. N-Y. Acad. Sci. 343, 1-16 (1980).

1085