Mastoparan, a wasp venom, activates platelets via pertussis toxin-sensitive GTP-binding proteins

Vol.

170,

No.

July

31, 1990

2, 1990

BIOCHEMICAL

RESEARCH

COMMUNICATIONS Pages

VIA

MASTOPARAN, PERTUSSIS

A WASP VENOM, TOXIN-SENSITIVE Yuki

Yukio Ozaki*, Masaaki Higashihara**, *Department **First

of

of

Yutaka Kariya***,

Laboratory Yamanashi,

Medicine,

Saga

Yatomi**, and Shoji

Medicine, Japan

Internal Medicine, of Tokyo, Tokyo

Internal

779-785

ACTIVATES PLATELETS GTP-BINDING PROTEINS

Matsumoto*, Toshitsugu

Clinical and Medical College,

Department of University

***Department Received

BIOPHYSICAL

AND

Yamanashi

Faculty ,Japan

Medical

Kume*

of

Medicine,

College,

Saga

,Japan

May 29, 1990

SUMMARY: Mastoparan induced limited release of serotonin from intact human platelets, while neither intracellular calcium ion elevation nor arachidonic acid mobilization was observed. Cytolysis induced by mastoparan was negligible in the concentration range that induced serotonin release. In digitoninpermeabilized cells ,++mastoparan induced Ca++-independent release arachidonic acid release. of serotonin and Ca -dependent Both serotonin release and arachidonic acid release were reduced by pertussis toxin, suggesting that platelet activation induced by mastoparan is mediated by GTP-binding proteins. 01990 kademic Press, Inc.

from

Mastoparan

is

wasp

(1).

venom

nulation,

it

It

activities. helical its

been is

binding

to

hydrolysis aspect

has

or

(2).

(5) neutrophils,

to

have

This

and

for

of

a wide

of

of

mechanism

functional

islets,

biologic an alphato

phosphatidylRecently, of

a

mastoparan-

GTP-binding have

degra-

contributes

A2 (4).

workers

pancreatic

cell

assumes

probably

mastoparan-induced and

mast

stimulation

the

isolated

range

which

involves

modifies

inhibits

report

phospholipase

Several

which

originally

property

(3).

which

toxin

peptide

proposed

G proteins. toxin

first

an amphiphilic

been

proteins

proteins

found

by purified

activation,

pertussis

the

calmodulin

induced

cells,

Since

configuration

choline new

has

a tetradecapeptide

regulatory

demonstrated

that

properties

of

activation

of

especially

that

G

mast of

0006-291X/90 $1.50 779

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

Vol.

170,

No.

2, 1990

intracellular

Cat+

Higashijima

fi

incorporated

igated

the

in of

our

paper

mastoparan

cell

types, has

release dealing

from

have its

not

platelets

mastoparan-induced we are

platelet

attempted

induced

by

activation

stimulates

effect fully

was

reported

G proteins

that

mastoparan

receptors

been

been

cell to

Furthermore,

agonist-liganded

of

COMMUNICATIONS

(6-8).

suggesting

by mimicking

of

RESEARCH

mastoparan

bilayers,

knowledge,

communication, activation

that

lipid

BIOPHYSICAL

mobilization

found

effects

a number

serotonin

induced

&

G proteins

While

AND

([Ca++]i)

into

activates

best

BIOCHEMICAL

(9).

extensively

invest-

on platelets,

to

evaluated; in

activation

the

the

limited very

first In

(1).

this

clarify

what

aspects

of

mastoparan,

and

whether

mastoparan-

via

G proteins.

is

MATERIALS

mediated

platelet

ANJ) METHODS

Mastoparan was obtained from Peptide Institute (Osaka, Japan). Pertussis toxin was purchased from List Biological Laboratories (CA, USA). Platelet-rich plasma was obtained from venous blood of healthy Platelets were isolated from plasma by centrifugation, donors. washed with a Hepes-Tyrode’s buffer containing 129 mM NaCl, 2.8 mM KCl, 0.8 mM KH2P04, 8.9 mM NaHC03, 0.8 mM MgCl 10 mM Hepes an resuspended in t i e same buffer and 5.5 mM glucose, (PH 7.2), at a cell concentration of 2 x 10I3 /ml, unless otherwise stated. Release of serotonin and arachidonic acid mobilization m asured with [3H]arachidonic acid-loaded cells and w”pt’z [ 8 Hlserotonin-loaded cells, respectively. Platelets were activated with various concentrations of mastoparan for 10 min at 37” c, and released label was expressed as percentage tothetotal radiolabel incorporated into platelets. Platelet [Ca++]i was measured with fura 2, a Ca++ -sensitive fluorescent dye, incorporated into cells. Fura 2 fluorescence was detected with dual-wave fluorescence spectrophotometry using a Hitachi F-2000 fluorescence spectrophotometer. Cell permeabilization was performed with digitonin. Briefly, platelets were suspended in a buffer containing 130 M KCl, 11.9 mM NaHC03, 0.42 mM NaH P04, 2 mM MgCl2, 5.6 mM glucose, and 10 mM Hepes (pH 7.4). 2 fter the addition of 1 mM EGTA, 1 ILM prostaglandin 12, and 20 Dg/ml digitonin, the cell suspension was incubated another 10 min at room temperature. Platelets were esuspended in the same buffer at a cell then washed once, and concentration of 2 x 10 B/ml. treatment: toxin was Pertussis toxin Pertussis preactivated with 20 mM dithiothreitol at 37” C for 30 min. Pertussis toxin (2.5 ,ug/ml) , 2 mM ATP and 0.2 mM NAD were added to the cell suspension of permeabilized platelets, and the mixture were incubated for 20 min at room temperature. Controls consisted of a permeabilized cell suspension with ATP and NAD but without pertussis toxin, and a non-permeabilized cell suspension with pertussis toxin, ATP and NAD. 780

Vol.

170,

No.

BIOCHEMICAL

2, 1990

AND

RESULTS Mastoparan release, Since

which a

far

abilized

was

not

which

membranes

bovine

adrenal

mine

release

upon

cell

with

may not

been

activation

concentration cannot

below

range

contrast

mastoparan

with

induced

cell the

neither

of

COMMUNICATIONS

described provide

to

induced

extracellular

serotonin

serotonin

Cat+

was

released

elsewhere an easy

(Fig. from

in

entry

for

in

upon

intact

intracellular

amount

of

suggesting

that

paper,

mastoparan. catechola-

cell

lysis,

platelets LDH

released

1). perme-

this

mastoparan-induced

be dependent

of the

to

by

However,

tested,

be attributed In

shown

release far

manner

cells,

(10).

mastoparan, or

is

chromaffin

has

negligible

affected

amount

RESEARCH

AND DISCUSSION

a dose-dependent

greater

cells,

platelet In

in

BIOPHYSICAL

not treated

was

almost

serotonin

in

serotonin

the

release

lysis. positive

effect

arachidonic

on acid

serotonin release

release, nor

[Ca++]i

30 % & ; u” m 2 2 2

01

0 Mastoparan

Figjre

1.

( u g/ml)

Serotonin

and LDH release

5 Mastoparan

10

20 (II

g/ml)

induced by mastoparan.

A [ Hlserotonin-loaded platelet suspension was incubated indicated concentration of mastoparan for 10 nin at platelet suspension was then rapidly centrifuged, resultant supernatant was assayed for released serotonin (Open circles, serotonin; closed circles, LDH) The presented as the means *SD of 5 experiments.

with C. and and data

37”

the The the LDH. are

Figure & Effect of Ca++ on serotonin release from permeabilized pjatelets induced by mastoparan. [ Hlserotonin-loaded platelets were permeabilized with digitonin and resuspended in a buffer containing either 10 DM Ca*+ or 1 mM EGTA. Mastoparan at the indicated concentration was added to the platelet suspension, and the mixture was incubated for 10 min at After rapid centrifugation, 37” c. the supernatant was assay for the content of released serotonin. (Open circles, Ca++ 10 ,uM; closed circles, EGTA 1 mM) The data are presented as the means +SD of 3 experiments.

781

40

Vol.

170,

No.

2, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

1 2.5

5 Mastoparan

10 ( p g/ml)

COMMUNICATIONS

I

x 20

40

Figure 3. Arachidonic acid mobilization from permeabilized induced by mastoparan. pJatelets [ Hlarachidonic acid-loaded platelets were permeabilized with digitonin and resuspended in a buffer containing either 100 UM Ca++ or 1 mM EGTA. Mastoparan at the indication concentration was added to the cell suspension, and the mixture was incubated for 10 min at 37” C. After incubation, the cell suspension was rapidly centrifuged, and the supernatant was assayed for the released arachidonic acid. (open circles, 100 PM Ca** ; closed circles, 1 mM EGTA) The data are presented as the means +SD of 3 experiments.

elevation

in

intact

[Ca+‘]i

elevation

lipase

C activation,

against

mastoparan

tion

pathway

dependent tion

(7,

A2

activation 11).

of

In stimulator U/ml

Ca++,

G proteins

It signal

which

AZ.

is

In

transducindependent

lack are

with

show G protein-

C upon mastoparan

that

be

may

contrast

neutrophils

platelets

digitonin-permeabilized of

serotonin

for

cells,

release,

As observed

release

suggesting

mastoparan

and

release,

stimula-

certain

linked

types

to

of

phospholipase

to be elucidated.

thrombin.

serotonin

release,

phospholipase

Whether

mastoparan-sensitive C awaits

cells

acid

a certain

of

phospho-

respectively.

phospholipase

mast

against

of arachidonic

serotonin

C or

intact

absence

stimulates

for

The absence

shown). evidence

activation,

selectively

phospholipase

not

indirect

and the

required

platelets,

(data

provides

phospholipase

that

of

platelets

comparable

with

intact

was unaffected that

serotonin

the

signal release

mastoparan

by

to

782

independent

effect

the

presence

transduction is

the

cells,

the

was a potent of

0.5

magnitude

of

or

absence

of

pathway

employed

by

of

(Fig.

Ca**

2).

Vol.

170,

No.

2, 1990

BIOCHEMICAL

Arachidonic

acid

platelets.

As

M

could

not in

the

(Fig.

In

release

is

requires

the

contrast

with

from

derived

from

presence in

on the

of

part

phospholipase

that

ILL

Thr. (+)

mastoparan

04

(-)

Thr.

(-)

(-)

Mast. (-)

C-1 (+I

0

Mast. (+)

5

Agonist IAP

U/ml

Ca++ acid

of

was

acid

cells

does not

elevate

(-) (6)

which

arachidonic

intact

1 Thr. (-)

Mast. (-)

acid be

may

[Ca”]i

C-1 (+)

Thr. (+)

Figure 4. Effect of pertussis toxin on serotonin release induced by massoparan or thrombin. [ Hlserotonin-loaded platelets were permeabilized with digitonin and further treated either with 2.5 Dg/ml pertussis of saline for 20 min. Platelets toxin or an equivalent volume were then activated either with 0.4 U/ml thrombin or with 20 fig/ml the reaction w s terminated and the mastoparan. After 10 min. supernatant was assayed for the released [ 8 Hlserotonin. The data (open columns, no are presented as the mean of 2 experiments. dotted columns, thrombin 0.4 U/ml: hatched columns, stimul~ator; mastoparan 20 fig/ml). Figure 5. Effect of pertussis toxin on arachidonic acid mobilization induced by mastoparan or thrombin. j3H]arachidonic acid-loaded platelets were permeabilized with digitonin and further treated either with 2.5 ug/ml pertussis Platelets toxin or an equivalent volume of saline for 20 min. were then activated either with 0.4 U/ml thrombin or with 20 .ag/ml mastoparan. After 10 rain, the reaction was terminated and the the released [3H]arachidonic acid. supernatant was assayed for The data are presented as the mean of 2 experiments thrombin 0.4 :yiE? columns, no stimulator; dotted columns, hatched columns, mastoparan 20 fig/ml). 783

a

release

arachidonic

of

n

Agonist IAP

0.4

A2 activity,

The absence

mastoparan-activated

cells.

by

arachidonic

lo-*

arachidonic

release,

a larger

Ca++.

ground

permeabilized

induced

serotonin

the

up to

massive

that

mastoparan-induced

permeabilized

Ca++alone

induced

to

human platelets,

mobiliza.tion explained

Ca++

COMMUNICATIONS

with

(12-13), acid

of

RESEARCH

evaluated

reported

presence

for

3).

also

comparable

In

prerequisite

BIOPHYSICAL

arachidonic

mobilization,

thrombin.

was

previously

liberate

Mastoparan acid

release

AND

Mast (+)

Vol.

170,

in

No.

2, 1990

intact

platelets:

activation signal

aroused

have concept

been

with

that

the

to

control

suggesting

that

the

G proteins

of serotonin

findings

of

suppressed

Kajiyama

arachidonic

acid

G proteins.

arachidonic

arachidonic

was

acid

suggesting arachidonic

(data

toxin

the

(Fig.

4).

successfully

the

with

permeabiliz-

induced with

with

cytoplasm

With

ROW

by

20

pertussis

mastoparan

fig/ml

toxin,

was

mediated

from

permeab-

4).

Thrombin-induced cells

into

of

from

stimulation

were

suppressed effect

cells

toxin

the

be

magn-

intact

thrombin

release

stimulatory

the

preincubated

G proteins.

remarkably

(Fig.

the

platelets toxin

to

different

without

that

need

from

upon

incubated

cell toxin

While

without

serotonin

of

on

cells

platelets

treated, also

induced

significantly

incubated

ADP-ribosylation

was

Ca++,

not

pertussis

effect

toxin.

release

less

This

pertussis

cytoplasm: the

types

proteins.

platelets,

with

was

cell

inhibitory

serotonin

cells

let

mastoparan

with

treatment

cells

to

thus

ilized

A2

activation

G proteins

the

substantiate

permeabilized

via

an

on several

the

human

into

released

findings

cells

and

GTP-binding

by

with

toxin

permeabilized

subsequent

COMMUNICATIONS

phospholipase

Ca++

ADP-ribosylates

up

the

toxin

These

of

via

digitonin-permeabilized

pertussis

of

mastoparan

mediated

thrombin-induced

shown),

presence

substantiated

prior

incubated

ed

be

taken

permeabilized

with

RESEARCH

picture

of

However,

readily

of

the

which

(5-9).

not

than

to

toxin

function

full

effects

shown

pertussis

BIOPHYSICAL

by mastoparan.

has

itude

both

stimulator-y

been

AND

the

may need

The

is

BIOCHEMICAL

et -A

al

acid by

pertussis

(13)

and

mobilization Similarly, release that acid

induced pertussis

induced

mobilization toxin, Nakashima by toxin

&

are

mobilization 784

in

5).

the in

the

(14) was

effectively

involved (Fig.

al.

thrombin

by mastoparan

G proteins

confirming

that

coupled blocked

presence mastoparan-

of

Vol.

170,

No.

2, 1990

Our and

findings

serotonin is

addition,

activate

pase

it

(most C.

particular thesis, the

is

direct

subsets

of

role

of

G proteins

in

activated

without

is

of

for

tools

to

binding

prove for

serotonin phospholi-

mastoparan

essential

by

selectively

activating

useful

platelet

may

pathways

demonstration

provide

mobilization

G proteins.

mastoparan

transduction

will

acid

platelets

that

G proteins

COMMUNICATIONS

toxin-sensitive

G proteins)

While

mastoparan

arachidonic

pertussis

suggested

probably

RESEARCH

permeabilized

by

signal

BIOPHYSICAL

that

from

mediated

certain

release

AND

demonstrate release

mastoparan In

BIOCHEMICAL

this

to hypo-

investigating

function.

REFERENCES 1. and

Hirai,Y., Kitada,

Yasuhara,T., Yoshida,H., Nakajima,T., Fujino,M., (1979) Chem. Pharm Bull. (Tokyo) 27, 1942-1944 Wakamatsu K Higashijima,T., Fujino,M., Nakajima,T., and iiyazawa,T. (i983) FEBS Lett. 162, 123-126 3. Cachia,P.J., Van Eyk,J.E., Ingraham,R.H., McCubbin,W.D., Kay,C.M., and Hodges,R.S. (1986) Biochemistry 25, 3553-3562 4. Angiolas,A., and Pisan0,J.J. (1983) J. Biol. Chem.258, 1369713702 5. Ohta, H., Okajima, F., and Ui, M. (1985) J. Biol. Chem. 260, 15771-15780 6. Higashijima, T., Uzu, S., Nakajima, T., and Miyazawa, T. (1987) In Peptide Chemistry 1986. T. Miyazawa, ed. Protein Research Foundation, Osaka, Japan, pp.75-78 7. Perianin, A., and Snyderman, R. (1989) J. Immunol. 143, 1669C.

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8. Yokokawa, N., Komatsu, M., Takeda, T., Aizawa, T., and Yamada, T. (1989) Biochem. Biophys. Res. Commun. 158, 712-716 9. Higashijima, T., Uzu, S., Nakajima, T., and Ross, E.M. (1988) J. Biol. Chem. 263, 6491-6494 10. Wilson, S.P. (1989) FEBS Lett. 247, 239-241 11. Okano, Y., Takagi, H., Tohmatsu, T., Nakashima, S., Kuroda, Y Saito, K., and Nozawa, Y. (1985) FEBS Lett. 188, 363-366 12: Nakashima, S., Suginuma, A., Matsui, A., Hattori, H., Sato, M Takenaka, A., and Nozawa, Y. (1989) Biochem. J. 259, 139-144 13: Kajiyama, Y., Murayama, T., and Nomura, Y. (1989) Arch. Biochem. Biophys. 274, 200-208 14. Nakashima, S., Hattori, H., Shirato, L., Takenaka, S., and Nozawa, Y. (1987) Biochem. Biophys. Res. Commun. 148, 971-978

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