Receptor binding activity and cytosolic free calcium response by synthetic endothelin analogs in cultured rat vascular smooth muscle cells

Vol. 160, No. 1, 1989

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

April 14, 1989

Pages

228-234

RECEPTOR BINDING ACTIVITY AND CYTOSOLIC FREE CALCIUM RESPONSE BY SYNTHETIC ENDOTHELIN ANALOGS IN CULTURED RAT VASCULAR SMOOTH MUSCLE CELLS Y. Hiratal*, H. Yoshimi2, T. Emoril, M. Shichiril, T.X. Natanabe3, S. Kumagaye3, K. Nakajima3, T. S. Sakakibara3 'Department

of Medicine,

2Department

Protein

Tokyo Medical Tokyo 113,Japan

F. Marumo', Kimura3 and

and Dental

University,

of Medicine, National Cardiovascular Center, Osaka 565,Japan 3Peptide Institute Research Foundation,

Inc., Osaka 562,

Japan

Received March 6, 1989 Summary: Using a variety of synthetic analogs of porcine endothelin (@ET), we have studied the effects of these analogs on receptor binding activity and cytosolic free Ca2+ concentrations ([Ca2+li) in cultured rat vascular smooth muscle cells (VSMC). Removal of C-terminal Trp21 residue, truncated derivatives pET(l-15) and (16-21), substitution of disulfide bond, Cys(3-II) or Cys(l-151, by all resulted in a complete loss of receptor binding Cys (km), activity and [Ca2+li response, while N-terminal elongation of LysArg residues, but not oxidation of Met7 residue decreased receptor binding activity and [Ca2+l. response. [ cys' -15 ,Cys3-IllpET was far more potent than [Cys 7 -+7 Cys3-15]pET in receptor binding and [Ca2+l i response. These data'indicate that the C-terminal TrpZ1 as well as the proper double cyclic structure formed by the intramolecular disulfide bonds of the PET molecule are essential for receptor binding and subsequent [Ca'+Ji increase in rat VSMC.

Porcine

endothelin

vasoconstrictor

peptide,

two intramolecular and sustained many species, (1 ).

(PET), comprises

disulfide

vasoconstriction of which

The cDNA cloning

a

effect for

linkages of

novel

21 amino-acid (1).

a variety

is dependent human (h)

and rat

* All correspondence should be addressed to: The Second Department of Internal Medicine, Dental University, Yushima l-5-45, Bunkyo-ku, 0006-291x/89$1.50 Copyright All rights

0 1989 by Academic Press, Inc. in any form reserved.

of reproduction

228

endothelium-derived residues

PET induces of blood

(r)

a potent

vessels

on extracellular

with

from Ca2+

ET has revealed

Yukio Hirata, M.D., Tokyo Medical and Tokyo 113, Japan.

Vol. 160, No. 1, 1989

that

hET

distinct these

BIOCHEMICAL

is

identical

from

PET (3). share

species

hydrophobic domains

to pET (2),

binding

have

the

for

(VSMC)

two disulfide

with

its

cytosolic

free

present

study

was

moieties

of

PET molecule

[Ca2+li synthetic

the

among

C-terminal

importance

presence

of these

cultured

rat

for

muscle

increase

(4).

Therefore,

characterize

essential

specific

smooth

a profound

([Ca2+]i) to

of

vascular

PET induces

designed

in

but

ET molecules

the

rat

Ca2+ concentration

response

similar,

receptor.

PET in cultured which

is

bonds and the

demonstrated

through

the

the

in common, suggesting

recently

sites

rET

and

Interestingly,

in interaction

We

cells

region

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

receptor

VSMC using

a

in the

structural binding

and

variety

of

PET analogs. MATERIALS AND METHODS

Peptides peptides were synthesized by solid-phase PET and related method and purified by ion-exchangechromatography on DEAEcellulose and reverse-phase HPLC as described The (5). homogeneity of the final products was confirmed by analytical HPLC and amino-acid analysis. Binding experiments Rat aortic VSMCs were cultured and used in the experiments as previously described (6). Binding study was performed essentially in the same manner as recently reported (4). In brief, confluent ( 5x105) cells were incubated at 37°C for 60 min with 2.5x10-" M 12SI-labeled-PET (Amersham Japan, specific activity: 2000 Ci/mmol) in the absence and presence of PET and related peptides. After completion, the cell-bound radioactivity was determined; specific binding was defined as total binding minus nonspecific binding in the presence of 4x1 0-7M unlabeled MPZHurement of [Ca2+]i Fluorescence of spectrofluorimetrically values were calculated al. (7).

fura-2-loaded cell measured as described by the formulas described

suspensions was (4)‘ and [CEt2+li by Grykiewicz et

RESULTS Competitive peptides inhibited approximate

is

binding

study

shown in Fig. the

binding

concentration

1.

of

by synthetic Unlabeled

1251-labeled-pET of half-maximal 229

PET

pET(l-21 to

rat

inhibition

and

related

) competitively VSMC with (IC50)

the of

Vol. 160, No. 1, 1969

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ENDOTHELIN Fig.

1. Competitive binding of 12SI-pET to rat VSMC by PET and related peptides. Confluent cells were incubated at 37'C for 60 min with 2.5~10-1~ M 1251-pET in the absence and presence of PET and peptides in concentrations as indicated. related Specific binding was 80% of total binding; each point is the mean of two experiments.

I

1

I

I

I

ENDOTHELIN Fig.

(M)

I

I

(M)

2. Competitive binding of 125X-pET to rat VSMC by PET analogs in rat VSMC. incubated at 37OC for 60 min with Confluent cells were 2.5x10-11 M 12SI-pET(.;n the absence and presence ,c$ and [Cysl-1',Cys3S]pET [Cysl-lS,Cys3-IllpET 82% in concentrations as indicated. Specific binding was each point is the mean of two experiments. of total binding; 230

Vol. 160, No. 1, 1989

3x10-"

was

while

M,

far

BIOCHEMICAL

less

In

[Cys(Acmj3r111-

and

biting

the

[Met(Oj71pET

pontent

respectively.

binding

than

of

in

[Lys-ArglpET

in

was

less

PET

a.

4

b.

Fig.

of

[Ca2+li

PET 4

”

IC50

about

[Lys-ArglpET

M and 2~10~~

10e10

pET(l-15),

1r151-pET in

of

and

were

loo-fold

displacing

pET(16-21),

ineffective

concentrations

in up

more

1251-pET

M,

to

inhi-

potent

from

M.

1O-7

than

its

binding

(10-T

M)

2). effects

increasing

potent

pET(l-20),

1251-pET

[Cy~l-l~,Cys3-~5]pET

The

PET with

[Cys(Acm)

was

(Fig.

was less

contrast,

[Cy~~-~~,Cys~-~~lpET

sites

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

[CyP5,

2 6 .-

266

ii-

152

g

104

Cyf-“IpET

PET

and

related

fura-2-loaded

peptides

VSMC are

potent,

but

[Met(0)7]pET

was

(Fig. almost

Met(O)‘-pET 4

Lys-Arg-pET 4

pET(l-flI)

compared

p7(1-21)

[Cys’-“, Cr3-‘l)pET

pET(l-is)

(Cys(Acm)3-“] &

~~(16-211

(Cys(Acm)‘-15) 4

3. Ca2+-furafluorescence by PET and related peptides in rat VSMC. Fura-2-loaded cell suspensions were challenged with 10m7 M each of (a) PET, [Lys-ArglpET and [Met(O171pET, (bl pETl1pET(l-20) following 21), pET(16-21) (c) [Cysl -1:,;;&;$;” ;by~$“~&;%~ 51;:: [Cys(A~m3r{~)lpET and , by )IpET as indicated arrows. Each panel shows a typical trace from the same cell preparations. Calculated values for [Ca2+li are shown on the ordinates. One-min interval is underlined. 231

on 3). as

Vol. 160, No. 1, 1969

potent

BIOCHEMICAL

as PET in increasing subsequent

and

pET(l-15),

sustained

nor

pET(l-21) treated

cells

whereas (Fig.

phases

(Fig.

with

(Fig.

of

both

initial

[Ca2+]i

increasing

increase

[Cys(Acm)3r111-

pET(l-20),

response,

[Ca’+li

although

in

[Ca2+li

[Cy~~-15,Cys3-~~]pET

3b).

transient

Neither

3a).

affected

capable

stimulating

on

[Ca2+li

pET(16-21)

was still

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

these

had greater

than

[Cpl-ll

and [Cys(Acm)1r151-pET

preeffect

,Cp3-15lpET,

were

ineffective

3~). DISCUSSION Using

clearly

of

demonstrates

novel PET

variety

a

the

endothelium-derived and related

with

those

increase

in

pET(16-21),

results,

failed

to

C-terminus

(Trp2l)

activity with

those

of

pET(l-15)

and

pET(lG-21)

orders

of magnitude

less

porcine

coronary

artery

together,

these

plays

an

stimulating While

data

of

residues

of

were

and

indicate

[Ca2+]i

increase,

of Met7

in

(Lys-Arg)

N-terminal resulted

complete

al.

residue

at

of

receptor

loss

These data

and ours

(5)

and pET(l-20) pET(l-21)

in

rat

pulmonary

artery.

C-terminal

interacting

although effect extension

in a marked

three

Taken

Trp2l ET

residue receptor,

vasoconstriction. slightly

on [Ca2+li of

are

constricting

with

and subsequent

the

in which

was

than

the

and

[Ca2+]i

and

even a single

(8)

232

that

pET(l-15)

binding

et

had stimulatory PET,

analogs,

response.

residue,

of

suggesting

ICa'+]i

that

a

correlated

almost

VSMC,

inactive

active

role

affinity, intact

removal

essential

oxidation

binding

receptor

Kimura

of

receptor-mediated.

affect

and subsequent

compatible

in rat

in a

study

The activities

binding

truncated

resulted

present

relationship PET.

by PET is

present

Surprisingly,

binding

response

induced

response.

the

structure/activity

in receptor

[Ca2+li

[Ca2+li

analogs,

vasoconstrictor

peptides

of

From the

that

synthetic

decreased similar

two basic

decrease

to

amino-acid in

receptor

BIOCHEMICAL

Vol. 160, No. 1, 1989

binding

activity

comparable

as well

to

One might

those

speculate

steric

hindrance

changes

of

the

Opening led

Acm

[Ca2+li

that

N-terminal

to

that

vasoconstrictive

loss

These each

structures

sites with

bonds at

Cys(3,ll)

of

or

by

activity

and

our

analogs

PET

for

Cys(l,l5)

with

Therefore,

important

are

receptor.

are consistent

(5).

conformational

binding

monocyclic

(5).

may somehow confer

its

of receptor

are

activities

and/or

interact

data

activity

Our results

elongation

active

the

a complete

observation

response.

vasoconstrictive

any disulfate

response.

ring

their

ET molecule

of

to

as [Ca2+li

of

on

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

two

recent had

no

intramolecular

receptor

binding

and

vasoconstriction. The present is

far

and

study

more potent [Ca2+li

Cys(3-11)

be

identical

although at the that

apamin

has

nor

Furthermore,

strongly by

two

suggested

critical

the positions has recently

on

1251-pET

the

double

disulfide for

linkages receptor

vasoconstriction. 233

in

with

apamin, bonds

We have

(IO).

binding

to high

has two disulfide

shown

[Ca2+li

(4),

observations). sarafotoxin

collectively, cyclic

the

Cys(l-15)

bee venom

been shown that

proper

their

been shown

(unpublished

Taken

of

sarafotoxins

to PET,

(5,ll).

intramolecular

bonds

In contrast,

has recently

that

Two disulfide

and Cys(3-15)

no effect

binding

with

snake venom

vasoconstriction it

is

of

in receptor consistent

at

location

Cys(l-II)

vasoconstrictor

Cys(3-11)

situated

homologous of

is

(5).

to PET (9).

structurally positions

potent

that

homology

response

which

of which

to

[Cysl-15,Cy~~-~'lpET

[Cy~l-~~,Cys~-~~lpET

of PET are (I),

sequence

shows that

activities

product

and

than

response,

vasoconstrictive natural

further

it

structure

between binding

is

Cys(l-15)

to

a is

formed and induce

Vol. 160, No. 1, 1969

It the

is

proper

essential

thus

BIOCHEMICAL

concluded

double for

receptor

that

cyclic binding

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

C-terminal

Trp2l

residue

and

structure

of

PET

molecule

are

and

[Ca2+]i

the

increase

in

rat

VSMC.

ACKNOWLEDGMENTS We thank Drs. M. Yanagisawa and Basic Medical Sciences, University discussion. This study was supported from the Ministry of Health and Welfare the Ministry of Education, Culture, and

T. Masaki, Institute of of Tsukuba, for helpful in part by Research Grants (62A-1, 63C-I) and from Science, Japan.

REFERENCES 1. 2. 3.

Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K. and Masaki, T. (1988) Nature 332, 411-415. Itoh, Y., Yanagisawa, M., Ohkubo, S., Kimura, C., Kosaka, T., Ishida, N., Mitsui, Y., Onda, H., Fujino, M. and Inoue, A., FEBS Lett. 231, 440-444. Masaki, T. (1988) Yanagisawa, M., Inoue, A., Ishikawa, T., Kasuya, Y., Kimura, S., Nakajima, K., Watanabe, T.X., Sakakibara, S * I Kumagaye, and Masaki, T. (1988) Proc. Natl. Acad. Sci. S Goto, K. vii.,

4.

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6964-6967.

Yoshimi, H., Watanabe, T.X., Hirata, Y., Takata, S., Kumagaye, S., Nakajima, K. and Sakakibara, S. (1988) Biochem. Biophys. Res. Commun. 154, 868-875. Kumagaye, S., Nakajima, K., Nishio, H., Kuroda, H., Watanabe, Masaki, T. and Sakakibara, S. (1988) In. T.X., Kimura, T., Peptide Chemistry ted. M.Ueki), Protein Research Foundation, Osaka (in press). Yoshimi, Hirata, Y., Tomita, M., H. and Ikeda, M. (1984) Biochem. Biophys. Res. Commun. 125, 562-568. Grykiewicz, G., Poenie, M. and Tsien, M. P. (1985) J. Biol. Chem. 260, 3440-3450. Kimura, S., Kasuya, Y., Sawamura, T., Shinmi, O., Sugita, Y., Yanagisawa, M., Goto, K. and Masaki, T. (1988) Biochem. Biophys. Res. Commun. (1988) 156, 1181-1186. Bdolah, A., Z. and Takasaki, C., Tamiya, N., Wollberg, Kochva, E. (1988) Toxicon 26, 543-548. Callewaert, G.L., Shipolini, R. and Vernon, C.A. (1968) FEBS Lett. 1, 111-113. Sokolovsky, M., Kochva, E., Wollberg, Kloog, Y., Ambar, I., 2. and Bdolah, A. (1988)Science 242, 268-270.

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