A molluscan neuropeptide related to the crustacean hormone, RPCH

Vol.

167,

No.

February

1, 1990

28,

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

1990

A MOLLUSCAN

NEUROPEPTIDE

RELATED

TO THE CRUSTACEAN

273-279

HORMONE,

RPCH

Yoshihiro Kuroki, Tomoko Kanda, Ichiro Kubota+, Yuko Fujisawa, Tetsuya Ikeda, Atsushi Miura, Yoshiharu Minamitakef, and Yojiro Muneoka" Physiological Sciences, +Suntory #Suntory Received

Laboratory, Faculty Hiroshima University, Bio-Pharma

Tech Center, Gunma 370-05,

Institute

January

for

of Integrated Arts and Hiroshima 730, Japan Chiyoda-machi, Japan

Biomedical

Research,

Ohra-gun,

Osaka

Japan

618,

22, 1990

A peptide that potentiates twitch contraction of the radula retractor muscle of the prosobranch mollusc Fusinus ferrugineus was isolated from the ganglia of the animal. Its primary structure is H-Ala-Pro-Gly-Trp-NH (APGWamide) closely related to the C-terminal tetrapeptide OZ the crustacean red-pigmentconcentrating hormone. APGWamide showed modulatory actions on contractions in various molluscan muscles. 01990 Academic Press, Inc.

A number AKH have have

been

strongly substance isolated of *To

of

been

neuropeptides isolated

found

in

from other

suggested

that

related

to

a RPCH-related

a prosobranch

mollusc,

whom correspondence

which

belong

arthropods,

phyla'.

but

However,

molluscan RPCH-AKH2. bioactive

ganglia In

the

tetrapeptide

Fusinus should

to

ferrugineus.

the none

family

of

of

such

Greenberg

et

possess

RPCH-

peptides al.

have

a peptidic

present

study,

from

the

We report

we

ganglia here

be addressed.

Abbreviations: ABRM, anterior byssus retractor muscle; ACh, adipokinetic hormone; APGWamide, H-Ala acetylcholine; AKH, locust seawater; FAB-MS, fast atom -Pro-Gly-Trp-NH2; ASW, artificial N-(2-hydroxyethyl)bombardment mass spectrometry; Hepes, HPLC, high performance piperazine-N'-(2-ethanesulfonic acid); liquid chromatography; 5-HT, serotonin; PTH, phenylthiohydantoin; TFA, red-pigment-concentrating hormone; RPCH, crustacean tris(hydroxymethyl)aminomethane. trifluoroacetic acid; Tris, 0006-291X/90 $1.50 273

Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in an.v form reserved.

Vol.

the

167,

No.

1, 1990

BIOCHEMICAL

purification,

activities

of

AND

structure the

RPCH-related MATERIALS

BIOPHYSICAL

determination molluscan

RESEARCH

and

COMMUNICATIONS

biological

neuropeptide.

AND METHODS

Animals---The animals used in the experiments are as follows: Euhadra congenita Fusinus ferrugineus (Prosobranchia), (Pulmonata) and Mytilus edulis (Bivalvia). Euhadra were collected in the campus of Hiroshima University. Fusinus and Mytilus were collected from Hiroshima Bay. Purification---The cerebral, suboesophageal and buccal of Fusinus. The acetone extract ganglia were excised from 1,100 of the ganglia was forced through C-18 cartridges (Waters SepPak). The retained material was eluted with methanol and gelThese filtrated with a column of Sephadex G-15 (26x400 mm). procedures were the same as those described previously3. The fractions showed four peaks of activities on twitch contractions of the radula retractor of Fusinus. However, each of the peaks seemed to be eluted partially overlapping with one or two other peaks. Therefore, we divided the active fractions into two The RPCH-related fractions 21-40 and fractions 41-58. wows, peptide was obtained from the latter group. The fractions of the latter group were pooled, concentrated and subjected to HPLC (Jasco TRI ROTAR VI) separation with a C-18 reversed-phase column (Tosoh ODS-80TM). The column was eluted with a 60-min linear gradient of O-60% acetonitrile in 0.1% TFA at pH 2.2. Three peaks of activities were obtained, twitchpotentiating, The active contractile and twitch-inhibiting peak. substance in the potentiating peak was then applied to a cationexchange column (Tosoh SP-5PW) and eluted with a 50-min linear gradient of O-O.5 M NaCl in 10 mM phosphate buffer at pH 6.8 Final purification was performed by applying the (Fig. IA). active material again to the C-18 reversed-phase column and eluting with a 30-min linear gradient of 15-20s acetonitrile in 0.1% TFA at pH 2.2 (Fig. IB). After each purification step, the bioactivity of each fraction was examined on train of twitch contractions of the isolated Fusinus radula retractor mounted in a chamber (2 ml). The methods of dissection, stimulation and recording from the muscle were the same as those described previously3. -The potentiating activity of the purified substance on the twitch contractions is shown in Fig. IC. Structure determination---The purified active substance was subjected to amino acid sequence analysis by automated Edman degradation with a gas-phase sequencer (Applied Biosystems 477A) coupled with a PTH-amino acid analyzer (Applied Biosystems 12OA), amino acid analysis (Hitachi L-8500) and FAB-MS analysis (JEOL JMS HX-TOO), and thus the probable structure of the substance was determined to be a tetrapeptide amide, APGWamide. The peptide was then synthesized by a manual solid-phase method followed by HF cleavage and HPLC purification. Its behavior on HPLC and bioactivity on the radula retractor were compared with those of the native peptide. Biological actions---Biological actions of APGWamide were examined on the following molluscan muscles: the radula retractor and protractor of Fusinus, the crop and the pharyngeal retractor of Euhadra, and the ABRM and the pedal retractor of Mytilus. These muscles, except the crop, were isolated and mounted in the same chamber used in the bioassay experiments. The methods of stimulation of the muscles and recording of tension from them 274

Vol. 167, No. 1, 1990

BIOCHEMICAL

0

20

40

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

hin)

Time

Fig. 1 HPLC purification of twitch-potentiating substance from the ganglia of Fusinus and bioactivity of the purified substance. A: the second step of the purification with a cation-exchange column. B: The third (final) step of the purification with a C-18 reversed-phase column. In A and B, bioactive peaks were coincident with the absorbance peaks indicated by arrows. c: potentiating action of the purified substance on train of twitches of the radula retractor of Fusinus. The train contr,actions were evoked by applying repetitive electrical pulses of stimulation (15 V, 2 msec, 0.2 Hz, 5 pulses) at 10 min intervals. The purified substance (PS) was applied to the muscle 8 min prior to the stimulation and washed out soon after it.

were also the same as those used in the bioassay experiments. APGWamide was applied to the muscles by replacing the solution in the chamber with the solution containing the peptide. The crop was excised by cutting at its both ends. The surrounding tissues were removed. The preparation was suspended in a 2-ml aerated organ bath. APGWamide was applied to the muscle by injecting the stock solution into the bath. Salines---The physiological saline for Mytilus muscles was ASW -of the following composition: 445 mM NaCl, 10 mM KCl, 10 mM CaC12, 55 mM MgC12 and 10 mM Tris-HCl; pH 7.8. For the saline of the Fusinus muscles, low-Mg2+ ASW (20 mM MgC12 ASW) was used to obtainlarge twitch contractions. This saline was made by replacing a part of MgCl2 in the normal ASW with osmotically equivalent NaCl. The physiological saline for the muscles of the land snail Euhadra was of the following composition: 120 mM NaCl, 4mM KCl, 0.9 mM CaC12, 2.5 mM MgC12, 1.5 mM glucose and 10 mM Hepes-NasOH; pH 7.5. RESULTS

The amino

determined acid

substance

in

the

were

Quantitative

sequence

as

amino

composition

normalizing

From

results,

whose

these structure

is

DISCUSSION

and

detected

acid

follows: acid

amino

AND

sequence

analysis

Ala65.3-Pro60

analysis

substance

H-Ala-Pro-Gly-Trp-OH 215

Pro, is

(pmol) of

of

the

each

purified

. .,-G~Y~~ . 3-Trp33.5.

showed the

on Ala=l: the

amount

0.8;

following Gly,

assumed or

its

amino acid

1.0; to

Ala,

1.0.

be a peptide

C-terminal

amide

Vol.

167,

No.

BIOCHEMICAL

1, 1990

derivative.

A molecular

MS

of

spectrum

the

the

ion

AND

BIOPHYSICAL

peak of

substance

429.1

in

was

RESEARCH

COMMUNICATIONS

(M+H)+

m/z

agreement

in the

with

FAB-

an amide at

C-terminus. The

at

synthesized

the

phase

APGWamide

identical column showed

elution

(Fig.

the a single

concluded

that

the

the

Trp-NH2).

The the

the

in both

of

total

yield

of

amino

acid

analysis

reversed-

of

of

the

the

cases dose

and

retractor

for

Thus,

we

Fusinus

peptide

is

to

the

from

the

related

is

(pGlu-Leu-Asn-Phe-Ser-Pro-GlyAPGWamide

calculated 3.01

was

nmol

from

1,100

animals.

0.03r

A

N.

A

c

,: ,,

oLL_lL L 0

n’;..:.. 20

/:’

N 1.’ /4

,S

.-.-

Timn

40

of

2C).

(Fig. the

eluted

mixture

radula

which

RPCH

C-18

between

of

structure

were

A

Fusinus

(APGWamide),

tetrapeptide

of

peak

identical

primary

C-terminal

result

in

almost

H-Ala-Pro-Gly-Trp-NH2

to

The relationship

action were

peptide

column.

absorbance

2A and 2B).

peptides

native

applied

cation-exchange

twitch-potentiating both

the

when

positions and

peptides

and

(min)

Fig. 2 Comparison of chromatographic properties and bioactivity of native peptide with those of synthetic APGWamide. A: reversed-phase chromatograms of the native (N) and synthetic (S) peptides and their mixture (N+S). B: cation-exchange chromatograms of the native (N) and synthetic (S) peptides and their mixture (N+S f . C: potentiating activities of the native (NJ and synthetic (S) peptides on total tension of 5 train twitches of the radula retractor of Fusinus. Each total tension is indicated as a percentage of the control total tension. The procedures for application of the peptides and recording of train twitches were the same as in Fig. IC. The concentrations of the native peptide were calculated from the result of amino acid analysis. 276

Vol.

167,

No.

BIOCHEMICAL

1, 1990

IO-‘M

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

APGWo

Fig. 3 Effects of APGWamide on some molluscan muscles. A: potentiating effect on train twitches of the radula retractor of Fusinus. B: inhibitory effect on train twitches of the radula protractor of Fusinus. The procedures in A and B were the same as in Fig. 1C. C: Inhibitory effect on spontaneous contractions of the crop of Euhadra. At downward arrow, the crop was washed with normal saline for 10 min. D: inhibitory effect on tetanic contraction of the ABRM of Mytilus. Tetanic contraction was evoked by applying repetitive electrical pulses of stimulation (15 V,, 3 msec, 10 Hz, for 5 set) at 10 min intervals. The other procedures were the same as in Fig. 1C. E: inhibitory effect on relaxation of catch tention in response to repetitive electrical pUlSeS of stimulation (15 V, 2 msec, 1 Hz, 15 pulses) in the ABRM of w:ilus. Catch tension was produced by applying ACh for 2 min

at 20 minintervals.

Although

APGWamide

contractions

in

the

inhibitory

action

thresholds

for M and

~xIO-'~

twitches,

even

by

In

of

M,

of

muscles,

the

not

only

muscles

the

crop

retractor

contractions were

(Fig.

of Euhadra of the

also

ABRM (Fig.

inhibited

by

the

of

the

ABRM was not

tension

were

not

M or higher

10v7

of

the

development 277

twitch

3D) and the

ACh contraction

contraction

the

were inhibited

inhibition

by

to

3C) and

the

potentiated

inhibit

the

contrast

for

but

tetanic

were approximately In

of

The

3A and B).

thresholds

M.

In the

(Fig.

showed an

peptide. of

Mytilus

3x1O-1o-1O-g

slightly

the

both

on twitch it

Fusinus,

respectively.

pharyngeal

Tetanic

of

and inhibition

in

M of

10d5

action

protractor

potentiation

the

retractor

these

radula

contractions

by APGWamide. pedal

a potentiating

retractor

contractions

Spontaneous contraction

the

5~10~~

ACh

affected

radula

in

the

showed

peptide.

were between inhibited

APGWamide.

ABRM, APGWamide was found but

also

relaxation

to

(Fig.

Vol.

167,

No.

I,

1990

BIOCHEMICAL

Therefore,

3D).

relaxation stimuli.

catch

The in

stimulation

(Fig.

in

response

relaxing

relaxing

M.

It

has

applied

been

to

relaxing

ABRM relax

nerves

to

the

suggest

that

has

by

also

been

transmitter

in

potentiates

tetanic

peak

tension

result

of

tested

5-HT

twitch

contractions

whereas

closely potent In molluscan

of

tetanic

of

that

RPCH of

retractor

the

protractor

to

Fusinus

muscles,

.

In

decrease might

found

a We

that

the

amine,

muscles

Yanagawa

this

be

by the

radula

a

in

The actions

the

is

5-HT

release.

inhibited

contractions

which

relaxing

that

5-HT and

release.

relaxing

the

The

on

ABRM

elements.

shown

Fusinus

of

the

the

of

potentiated.

twitch

10

of

APGWamide

peptide

contractions

twitch-inhibiting summary,

in

were

thomasiana

5-HT6r7.

nerve

ABRM7!'.

of

5-HT

intramuscular

from

been

the

were

of

5-HT

release

has

the

of

potentiates

Rapana

related

of muscles

inhibition

of

the

pulses

experiments

contraction

radula

twitch

the

inhibit

contraction

of

approximately

the

intramuscular

It

the

on the

be results

retractor

of

to

action

the

the

ABRM8.

the

on

on

catch-

inhibition

transmitter

release

suggested

inhibitory

those

APGWamide

shown

of

in

and

was

of

relaxation

electrical

relaxing

the

pulses

5-HT,

stimulating

by

inhibits

the

the

repetitive

catch

acting

as

stimulation

that

on

on the

such

on

relaxing

effect

no effect

for

obtained

APGWamide

terminals

showed

release

results

various

electrical

threshold

suggested

APGWamide

inhibitory

electrical

the

Therefore,

nerve

The

to

repetitive

it

COMMUNICATIONS

of

response

monoamines4,

by repeititve

5x10-"

RPCH

but

RESEARCH

effect

potent

to

3E),

peptide5.

relaxation

in

showed

response

to

the

tension

peptide

relaxation

BIOPHYSICAL

examined

we

of

AND

et of

muscle,

might

al.

have

the

prosobranch 5-HT

of

radula mollusc

is

also

a

agent".

APGWamide further,

is

structurally both 278

of

related the

peptides

to seem

RPCH.

In

to

show

Vol.

167,

NON. 1, 1990

BIOCHEMICAL

qualitatively

similar

peptides of

5-HT

on

the

AND

BIOPHYSICAL

actions.

muscles

of

Some

may be

RESEARCH

brought

the

about

COMMUNICATIONS

actions through

of

the

inhibition

release. ACKNOWLEDGMENTS

The Y.

authors

wish

to

express

and

Dr.

T.

Shimonishi

spectroscopic

their Takao

sincere

thanks

(Osaka

to

University)

Professor for

mass

measurement. REFERENCES

1. 2. 3.

Penzlin, H. (1989) Naturwissenschaften 76, 243-252. Greenberg, M. J., Rao, K. R., Lehman, H. K., Price, D. A. and Doble, K. E. (1985) J. exp. Zool. 233, 337-346. Kanda, T., Takabatake, I., Fujisawa, Y., Ikeda, T., Muneoka, M. (1989) Hiroshima J. Med. Sci. 38, 106Y. and Kobayashi, 116.

4. 5.

Twarog, B. M. and Cole, R. 46A, 831-835. Hirata, T., Kubota, I., Shimamoto, N. and Muneoka,

A.

Comp.

(1973)

Takabatake, Y. (1987)

Biochem.

I., Brain

Physiol.

Kawahara, Res. 422,

A., 374-

376. 6. 7.

Cambridge, G. W., Holgate, J. A. Phylsiol. Lond. 148, 451-464. York, B. and Twarog, B. M. (1973)

and Sharp, Comp.

J.

A.

(1959)

J.

Biochem.Physiol.44A,

423-430. 8. 9.

Saitoh, 389-395. Muneoka, 73C,

10. 11.

149-l

Yanagawa, Kobayashi Muneoka, 65C,

H. and Muneoka, Y.

and

Kamura,

Y.

(1986)

M.

(1982)

Hiroshima Comp.

J.

Med.

Biochem.

Sci.

35,

Physiol.

56.

M., Fujiwara, M., Takabatake, I., Muneoka, Y. and M. (1988) Comp. Biochem. Physiol. 9OC, 73-77. Y. and Kobayashi, M. (1980) Comp. Biochem. Physiol.

81-86.

279