Vol.

183,

March

No.

2, 1992

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages 849-855

16, 1992

INTERACTION AND

BETWEEN

ENDOTHELIN-1

ENDOTHELIUM-DERIVED

ON CA 2+

MOBILIZATION

Okishio,

Shigenori

Miki

and

Chemistry

Research

Chemical

February

5,

RAT

FACTORS,

VASCULAR

Ohkawa,

Koichi

Yuzo

S-NITROSOTHIOLS,

SMOOTH

MUSCLE

CELLS

Ichimori

Kondo

Laboratories,

Takeda

Received

RELAXING IN

Research

Industries,

and

Ltd.,

Development

Osaka

Division,

532,

JAPAN

1992

SUMMARY: S-Nitrosothiols (S-nitrosocysteine, S-nitrosoglutathione and Snitroso-N-acetylpenicillamine), which belong to the group of endotheliumcaused decreases of cytosolic free CaZ+ derived relaxing factors (EDRFs), concentrations ([Caz+],) in cultured rat vascular smooth muscle cells endothelin-1 (ET-1)-induced sustained increase of [Ca*+], in (VSMCs) . The rat VSMCs was completely abolished by preaddition of at least an equal molar quantity of S-nitrosocysteine (Cys-SNO). Also exposure of VSMCs to a mixture of Cys-SNO and ET-I at the same time resulted in the transient increase only. These results suggest that S-nitrosothiols may have no significant effect on ET-l-induced CaZ+ release from intracellular stores via inositol production but do affect Ca2+ influx 1,4,5-triphosphate through Ca2 + 0 1992 Academic Pre55, Inc. channels in the plasma membrane.

The

role

of

endothelial

vasoconstriction

(3-5)

Endothelin-1

(ET-l)

factors of

(EDCFs). cultured

Myers

for

but

et

potent

al.

l-induced

in et

al.

(12)

and rat

vascular

vasodilation

that

well

from (6).

the

ET-l

the

most

known.

supernatant is

relaxing of

and

contracting

purified

cells

and

is

endothelium-derived and

one

(10,ll)

(1 ,2)

substances

endothelium-derived

proposed

a

likely

factors

EDRFs,

(EDRFs)

S-nitrosothiol,

likely

accounts

is in

large

a

part

relaxations. and

relative

regulation and

contractions.

nitrosothiols cultured

the

potent

isolated endothelial

of

vasodilator

interaction

important Ltischer

the

aortic

endothelium-dependent The

of

mediating

vasoactive

recently

antagonist

(1,7-9).

in

releasing

one

was

porcine

physiological

labile

by is

It

cells

of

Vanhoutte

on smooth

local et

However, ET-1

potencies blood a1.113)

direct

cytosolic muscle

of flow

cells

in

reported evidence

free

EDCF

Ca2+ (VSMCs)

and health

that to

show

EDRF the

concentrations has

apparently

EDRF

may

and

disease.

inhibits effect ([Ca2+] never

be

ETof

S-

>)

in been

reported.

849

All

Copyright 0 1992 rights qf reproductkm

0006-291X/92 $1.50 by Academic Press, Irw. in any form reserved.

Vol.

183,

No.

In in

2, 1992

this

rat

BIOCHEMICALAND

study,

VSMCs

we

investigated

using

Furthermore,

the

mobilization

in

the

the

calcium

mechanisms these

BIOPHYSICALRESEARCH

by

cells

effect

S-nitrosothiols

sensitive

which

were

of

COMMUNICATIONS

on

fluorescent

S-nitrosothiols

[Caz+ll

probe and

ET-l

fura-2.

affect

Ca*+

studied.

MATERIALS

AND

METHODS

Measurement of [Caa’], : Ca*+-furafluorescence on a glass cover slide was measured at 37 “c previously described (14,15), and [Ca*+], values to the method of Grynkiewicz et al. (17).

in

from 3.2 x lo5 a spectrofluorimeter were calculated

rat

VSMCs as according

Effect of S-nitrosocysteine on Ca2+ mobilization induced by endothelin-1 : A glass cover slide coated with 3.2 x 10 5 furaloaded VSMCs was inserted diagonally into a quartz cuvette that contained 2.5 ml HEPES-buffered physiological salt solution (PSS : 140 mM NaCl, 5 mM KCI, 1 mM MgSO, , 1 mM Naz HPOl , 1 mM CaCl,, 25 mM glucose, 25 mM HEPES), pH 7.2, supplemented with 0.05 % bovine serum albumin. The cells were incubated with S-nitrosocysteine (Cys-SNO, 10mio, 10mg, 10m8, 1O-7 or 10e6 M) (18) for 10 min before application of ET-l (10m9, lo-@ or 1O-7 M). Rat VSMCs were also pretreated for various times (1, 3, 5, 10 and 20 min) with Cys-SNO (10m7, 1Oe8 or 10m9 M) before application of ET-l (lOma or 10m9 M). Effect of the mobilization : (10e9, 1OmB and was added to rat

mixture Cys-SNO 10e7 M) VSMCs.

Effect of endothelin-1 The cells were incubated application of Cys-SNO pretreated for various before application of

of

S-nitrosocysteine (lo-lo, 10m9, were previously

and endothelin-1 10m7 and 10m6 in tubes. Then

lo-*, mixed

on

Ca2+ mobilization with ET-l (10e9, (lo-*, 1O-7, 10m6 times (I, 3, 5, 10." M Cys-SNO.

on Ca2+ M) and ET-l each mixture

induced by S-nitrosocysteine : lo-* or 10m7 M) for 10 min before or 10e5 M). Rat VSMCs were also 10 and 20 min) with 1O-B M ET-l

Chemicals : Endothelin-1 was purchased from Peptide Institute, Inc. S-nitrosoglutathione (Glu-SNO) (18,19) and S(Osaka, Japan). Cys-SNO (18)) nitroso-N-acetylpenicillamine (Ac-Pen-SNO) (18) were synthesized by Takeda Ionomycin was purchased from Calbiochem (La Jolla, CA, Chemical Industries. U.S.A.). Glycoletherdiamine-N,N,N’ ,N’-tetraacetic acid and furaacetoxymethyl ester were purchased from Dojin Chemical (Kumamoto, Japan). All other reagents were of the highest grade commercially available.

RESULTS Addition rat

of

VSMCs

level

led

within for

lasting incubation

S-nitrosocysteine

to

a rapid

15

set,

5

SNO)

also

VSMCs (Glu-SNO)

induced

Cys-SNO

(10m9-10m5

changes

in

rat

a decrease M),

VSMCs

in

Glu-SNO [Caz+],;

1A with

one

[Caz+],

a subsequent

Figure

rat

nitrosoglutathione

decrease with

min.

of

(Cys-SNO),

. The

illustrates

in

S-nitrosothiols,

a

of

typical the

to

reached

phase

Among

a minimum

lowered

[Ca2+]

response

[Ca2’],

.

compounds

850

M)

Figure and

S(Ac-Pen-

2

depicts

Ac-Pen-SNO

cause

I

during

S-nitrosothiols,

S-nitroso-N-acetylpenicillamine

(10m7-10m4 These

the [Caz’],

sustained

Cys-SNO. and

of

dose-dependent

the

effects

(10m6-10m4

of M)

decreases

on

Vol.

183,

No.

BIOCHEMICAL

2, 1992

AND

I

10’

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

-

M

Cys-SNO

IO*

1 rmn

M

ET-1

1 ml”

Fig.1. Ca?+-furafluorescence by 10~” M S-nitrosocysteine (Cys-SNO) (panel A) and 10~’ M endothelln-1 (ET-l) (panel 8) in rat VSMCs. The trace reflects changes in [Ca*+], as evaluated from the changes in fluorescence of fura-2-loaded rat VSMCs cultured drrectly on glass cover slrdes. Each trace 1s representative of three similar experiments. Values of [Can+], are shown on the ordinate.

in which

[Ca’*]

I.

The

minimum

decreases

Glu-SNO

Cys-SNO,

in

[Gas’],

been

shown

could

be

and

detected

Ac-Pen-SNO were

concentrations

1Oms

M,

10-s

for

M and

10m5

VSMCs

[Cae+],

followed

by

M,

respectively. ET-l that sharp the

is

has

characterized

decline maximum

to and

to by

the lasted

cause a

sustained for

a two

phase,

9

increase

(15-20

-10-15

10

phase

transient

which min

6

set) was

(Fig.lB)

7

S-nitrosothiol

in

rat

increase

approximately

20

percent

(14,15).

6

5

4

3

(-log M)

Effect of S-nitrosocysteine (O), Fig.2. S-nitrosoglutathione (A) and Snitroso-N-acetylpenicillamine (U) on [Caz+lX in rat VSMCs as a function of concentration. [Cae+], decrease is the value obtained from fura-2-loaded rat VSMCs cultured directly on glass cover slides, following exposure to various doses of S-nitrosothrols as described in Methods. Values are mean k S.E. from 3 experiments.

8.51

a of

Vol.

183,

No.

2, 1992

BIOCHEMICAL

AND

t 10’

M

BIOPHYSICAL

t 10’

Cys-SNO

RESEARCH

M

COMMUNICATIONS

ET-1

0

10’h

10’

Cys-SNO

M

+ 1O’M

ET-1

lo6

ET-1

M

Cys-SNO

Fix. 3. Interaction between S-nitrosocysteine (Cys-SNO) and endothelin-1 (ET-l) in rat VSMCs [CaZ+], . (A) Effect of lo-’ M Cys-SNO on the lo-8 M ETl-induced [Cap*] I increase in rat VSMCs. (B) Effect of the mixture of 10.’ M Cys-SNO and lOen M ET-1 on the [Caz+], of rat VSMCs. (C) Effect of IO-8 M ET-1 on the 10.” M Cys-SNO-induced [Caz+] I decrease in rat VSMCs. The trace reflects changes in [Ca*+], as evaluated from the changes in fluorescence of fura-Z-loaded rat VSMCs cultured directly on glass cover slides. Each trace is representative of three similar experiments. Values of [Caz’], are shown on the ordinate.

Rat

VSMCs

contained were

10m7 then

but

were

the to

in

both of

the

[Caz+]

10-‘“-lO-”

M was

quantity

of

of

Cys-SNO (Fig.

a

completely

only

The

of

10mg

of

M Cys-SNO

preaddition

of or

at

M ET-l 1O-8

I and in

the also

1O-8

the

increase of

of time

pretreatment

and

Tables M ET-I

sustained

by

M ET-l increase

same

However,

1O-g-1O-7

ET-l-induced

the

addition

increases.

by

at

that

lo-*

transient exposure

M ET-l

before

sustained

abolished

to

the

(Fig.SB).

min

induced

Cys-SNO.

10-a

PSS

response

induced Interestingly,

mixture

and

in

[Ca2+li

and

10

HEPES-buffered

2 list

the

presence in

least

M ET-l

an

rat

of VSMCs

equal

molar

Cys-SNO. we rat

after

VSMCs the

examined to

ET-I

application

of

the

effect

of

adding

on

changes

in

their

lo-

*

M ET-l

Cys-SNO [Ca2+],

produced

10 .

min

after

Adding

a decrease

in

10m6

M

[Ca*+)

,

3C) . The

mobilization in

with

in

ET-l

(Fig.3A).

for

transient

min

in

increase

1 elevation

Furthermore, exposure

changes

M Cys-SNO

M Cys-SNO

treatment

produced

[Ca*+l x

10m7

transient

10

experiment,

increase of

10es

simultaneous

levels

this

sustained

the

with

for

The

In

a mixture

resulted cells

M Cys-SNO.

measured.

not

cells

incubated

a treatment

effect

of was

also

treatment examined.

time-independent

time

(1,

3,

Qualitatively manner

10

5, similar

(data

852

not

shown).

and results

20

min)

on

Ca2+

were

obtained

Vol.

183,

No.

2, 1992

Table

BIOCHEMICAL

Effect of transient

1.

induced ET-l

AND

S-nitrosocysteine and sustained

Extent Transient

of

[Ca*+],

increase

420 270

t i

1lOt 110 625 290 2801 210 1100 35Oi 340 320

10-9

1Om8 10-7 0 10~8 10 7 10-e

10-T

increase increase

(nM)

0 10-'0 10-g 10-a 0

10-a

COMMUNICATIONS

endothelin-1 (ET-I)in rat VSMCs

Sustained

CM)

9

IO-

RESEARCH

(Cys-SNO) on increases [Caz+lX

Cys-SNO

0.0

BIOPHYSICAL

10 8

12Ot 5Oi

6 6 0 0

6 t 13 x 50 t 13 8 f 28 t 60 8 I 14 i 6

175 5oi

t

14 10

0 0 240 t 30+ 20? 0

Furaloaded VSMCs (3.2 x 105) were pretreated min before application of various doses (10e9, Values are means i S.E. from 3 experiments.

with

18 3 4

Cys-SNO at 37 C for 10 and 10m7 M) of ET-l.

1Om8

DISCUSSION The factors in

present known

are

releasing

NO

been

the

shows have

VSMCs

(Figs.lA

labile

but

from

effect

clearly

S-nitrosothiols

rat

cultured

nitrosothiols

showing

study as

2).

and potently

endothelium of

that

endothelium-derived

a potent

effect

Myers

et

vasodilate

cells on

decreasing

al.

suggested

muscle

smooth However,

(10,ll).

S-nitrosothiols

relaxing

of

VSMCs

direct

[Gas+],

has

[Gas+]> that

S-

tissue

by

evidence

apparently

never

reported.

Table

2.

Effect of endothelin-1

ET-l

the (ET-l)

mixture of S-nitrosocysteine on the [Caz+lI in rat

Cys-SNO

Extent Transient

CM)

CM)

10-g

lo-

'0

O-8 0-g o- 8 o- 7

lo-

7

[CaZ+],

and

increase

Sustained

increase

(*M)

o- 9

10-a

of

increase

(Cys-SNO) VSMCs

O-8 o- 7 o- 6

Each mixture of Cys-SNO and ET-l are means VSMCs at 37 'c. Values

360 160 150f 480 280 240 450 430 420

F 18 I 10 9 i- 10 t 11 i 20 i 13 t 13 i 5

was administered to 3.2 t S.E. from 3 experiments.

853

60?

2

0 0 6Oi

4 0 0

6Oi

2 0 0

x

105

fura-

loaded

Vol.

183,

No.

2, 1992

ET-i

causes

both

[Ca*+],

(Fig.lB)

[Ca2+l

was

completely

of

Cys-SNO,

x

quantity remained with

of

to

less

than

was

The

of

and

by

the

induced

and

explanation

may the

than

Cys-SNO

VSMCs

were

increase

the

various

rat

ET-1

mobilization ratios

with

added

VSMCs to

molar

pretreated

subsequently

VSMCs molar

respect

Thus by

rat

of

with

VSMCs

equal

transient

Cys-SNO

increases.

rat

an

pretreatment

of

the

in

least the

rat

of

ET-l

Cys-SNO,

at

a

a decrease

(Fig.3C).

precise come

contrast,

differently

when

ratio

at

but

sustained

affected

by the

In

in

increase of

concentration

transient

molar

Al though

sustained

preaddition

1,2).

molar

COMMUNICATIONS

increase

S-nitrosothiols,

Tables

equal

RESEARCH

sustained

ET-l-induced

Furthermore,

equal

through

a

qualitatively

Cys-SNO.

still

and

one

the

is

ET-l

transient

abolished

an

both

[Ca”].

BIOPHYSICAL

a

3A,3B

than

produced

AND

(14-16).

(Fig. less

BIOCHEMICAL

reasons

from

for

these

understanding

voltage-dependent

phenomena

the

are

balance

Ca2+-channels

and

unknown,

between

CaZ+

an

CaZ+

efflux

influx

through

the

Caz+-pump. ET-I from

has

been

shown

intracellular

and

CaZ7

plasma

influx

(a

membrane

and

GMP

by

smooth

(23). Under

an

ET-1

in

C.

is

inhibited

not

ET-l recent

exposed

by logical

be

used

may

be

to

effective

most

et

that

antagonizing

by

from

al.

(12)

human

influx,

a potent

stores

consistent

on a

the

ET-l-induced

854

effects

be

conclusions

has

which

is

such

by a

ET-Ialso

been

apparently (25,26).

indicates

IP,

from

inhibits

increase

vasocontrictions.

to induced

ET-l

study

a

protein

response

EDRF

aorta,

to

C or in

of

related

that

antagonist This

neutralized

veins.

sustained

endogenous

contractions.

be

increase with

and

K’

efflux.

blunted

also

transient

describing

effect

Ca2+

may

The

CaZ+

arteries

Ca2+

phospholipase

the

[Caz+], studies

Ca2’-activated

ET-1

may

messenger,

cause

in recent

induce

efflux.

cyclic

then

lowering

of

soluble of

could

More

and

into

catalyzed of

stimulation

rapid

induced

is

inotropic

sustained

a

be

group

levels

probability

influx

Ca2+

and

(21,22).

open

Therefore,

normal

in

to

Ca2+-pump

of

explanation

increase

in

the

Alternatively,

heme

and

S-nitrosothiol

release

Liischer

therefore limit

lead

second

This

a positive an

an the

(24).

in

have

in

membrane

(11).

the

cGMP

Ca2+

cGMP

contraction to

the

Ca2+

to

the by

by

(IP,) channels

spontaneous to

pathways

activate

of

be

binds

S-nitrosothiol-induced

remain.

mediated

not

and

conditions,

report

induced

seems

also

However,

would

shown

enhance

can

effectiveness

kinase

increase)

plasma

NO

activation

biochemical

may

VSMCs

decreased

Ca2+

the

liberate

NO

enzyme

relaxation

NO

sized

transient

through

intracellular

This

these

equal

(a

1,4,5-triphosphate

through

to might

causes

muscle

that

channels

diffuse

(‘20).

Elevated

various

indicated

by

This

stimulating

to

membranes

formation.

vascular

shown

S-nitrosothiols

cyclase.

(cGMP)

release inositol

possibly

decompose

vascular

guanylate

Ca2+ by

increase)

were

of

external

both

mediated

.

spontaneously

denitrosation at

cause

stores

sustained

(16)

S-Nitrosothiols VSMCs

to

Ca*+

as

EDRF that

It may EDRF

Vol.

183,

No.

BIOCHEMICAL

2, 1992

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

ACKNOWLEDGMENTS We express our deep appreciation to Dr. and support throughout this work. Thanks are gift of S-nitroso-N-acetylpenicillamine the generous Motsenbocker for reviewing this manuscript.

S. also

Terao due

for his encouragement to Mr. S. Fukumoto and to Dr.

for M.

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Interaction between endothelium-derived relaxing factors, S-nitrosothiols, and endothelin-1 on Ca2+ mobilization in rat vascular smooth muscle cells.

S-Nitrosothiols (S-nitrosocysteine, S-nitrosoglutathione and S-nitroso-N-acetylpenicillamine), which belong to the group of endothelium-derived relaxi...
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