Vol.

89,

No.

2, 1979

July

27, 1979

BIOCHEMICAL

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

MODULATION

OF

ACTIVITIES

BY

RAT

MYOMETRIAL

SODIUM

Denis

Institut

AND

Leiber

and

Laboratoire d’Endocrinologie de Biochimie, Universite

Received

May

11,197

GUANYLATE

NITROPRUSSIDE FATTY ACIDS

CYCLASE

AND

Simone

598-606

UNSATURATED

Harbon

et Regulations Paris-Sud,

cellulaires 91405-Orsay

“LA Cedex,

272”, France

9

SUMMARY : Guanylate cyclase activities are present in both soluble and particulate fractions of rat myometrial extract. Triton, slightly stimulated the soluble (50 %) while markedly increasing (1000%; the particulate activity. Both fractions appear to be regulated independently. Predominantly, the soluble form was activated by sodium nitroprusside, involving interactions with SHgroups. On the other hand, the particulate form was stimulated by a series of unsaturated fatty acids and their hydroperoxides. The latter activation appears to result from direct hydrophobic effects rather than peroxide or free radical generation. INTRODUCTION:

In

physiological fective

in

does

other

cGMP

where

stimulation

not

xylamine

lead

to

reactions

may

activate

diverse

involving

the

hydroxyl

radicals

fatty

acids

has,

in

enzyme free

Thus,

be

related SNP,

cases,

(7,8),

it

has

formation

Abbreviations BHA, butylated

to

been also

soluble

sodium

two

direct

@ 1979 by Acudemic Press. Inc. of rqwoduction in anyform reserved.

the

of hydro-

different nitroprusside;

598

guanylate

radical sodium

a common

or

activation

pathway

ability

of various fractions

interactions

withthe

result

activation DTT,

by

particulate

could

systems,

azide

activation

the

hydrophobic that

free

cyclase

from

0006-291X/79/140598-09$01.00/0 Copyright All rights

Guanylate

cyclase

in

On

of

and via

Although

and,

an

influxes

presence

or

(6).

to

Ca+2

modulation

apparently (4).

suggested

used are: SNP, hydroxyanisole.

the

nitrosamines

been

(6)

that

described

ascribed

to be

content.

oxidation-reduction

oxide

guanylate

in

or

inef-

(1).

cyclases

been

cGMP

of

totally

appeared (1, 2)

enhanced

carcinogenic

also

tissue

indicates

of nitric

stimulate

some

myometrium

in

(SNP)

a series were

receptors

markedly

presently

formation

to

radical

were

guanylate

has

rat

increases

evidence

(3-6).

The

nitroprusside

Current activity

(1).

myometrium,

acetylcholine

of muscarinic

levels

sodium

rat

including

levels

consistent

cGMP or

cyclase

effecters

altering

hand,

may

oestrogen-dominated

contractile

exception (1)

the

dithiothreitol;

from of

Vol.

89,

No.

BIOCHEMICAL

2, 1979

guanylat’e

cyclase

oxidation

to

peroxides

The

present

mechani,sms guanylate

by

unsaturated

underlying cyclase

fatty

to be paper

AND

BIOPHYSICAL

acids

effective

(9,

reports modulation

RESEARCH

appeared

to

COMMUNICATIONS

require

their

10).

on

a study

designed

of

soluble

and

to

evaluate

particulate

possible

myometrial

activities.

EXPERIMENTAL

PROCEDURE:

- Preparation of tissue and assay of guanylate cyclase activity: Uteri were obtained from immature oestrogen-pretreated rats an myometrial strips were prepared free of endometrium as previously described (11). All experiments were carried out at 0-4°C. Myometrial strips were homogenized with an LJltra Turrax homogenizer in 10 volumes of cold buffer consisting of 0. 25 M sucrose, 5 mM Tris-HCl buffer pH 7. 5, 1 mM EDTA and centrifuged at 700 x g for 5 min. to eliminate cell debris. The pooled 700 x g supernatant fractions constitute the “crude extract” which was further centrifuged at 105. 000 x g for 1 h in order to obtain the particulate and the soluble cell fraction. The pellet was resuspended in a volume of buffer equal to that of the original homogenate. Guanylate cyclase activity was assayed by a modification of the method of Mittal et al. (4). The standard reaction mixture contained 50 mM Tris HCl buffer pH 7. 5, 0. 5 mM isobutylmethylxanthine, 550 pg creatine 1 mM GTP, 1 mM MnC12 in a phosphate, 33 pg creatine phosphokinase, final volu.me of 150 ~1. Various agents to be tested were included as indicated. The reaction was initiated by the addition of lo-20 pl of the supernatant or particulate fraction (15-30 pg of protein) and incubation was carried out at 37” for 10 min unless otherwise indicated Reactions were terminated by the addition of 1. 35 ml of 50 mM sodium acetate buffer pH 4. 0 and heated for 3 min at 90”. cGMP formed was determined in duplicate without purification by the radioimmuno assay ( 12) as modified by Cailla et al. (13). Protein was determined by the Lowry method (14). Hydroperoxy-derivatives arachidonic, linoleic and y-linolenic acids were obtained by catalytic oxygenation Iof the corresponding fatty acid using Soy bean lipoxygenase (15). The reaction was carried out at room temperature in 0. 5 ml of Tris HCl buffer pH 8. 0 with 600 PM fatty acid and 25 pl of lipoxygenase (5000 u). Measurement of the absorbance at 238 nm indicated that oxygenation was complete between 5 and 10 min. Material: (Serva) creatine SNP, cGMP Pasteur) Institut Merck

3-isobutyl-1 -methyl xanthine (Aldrich), Soy bean lipoxygenase the various fatty acids (Nuchek Prep Inc. ), Creatine phosphate and phosphokinase (Boehringer Mannheim), 2’-0-succin 1 cGMP, 1y25 I-succinyl DTT, N-ethylmaleimide, BHA and thiourea (Sigma), tyrosine methylester was prepared at URIA, (Dr. F. Dray, Institut and the antibody for radioimmuno-assay of cGMP was from Pasteur Production, France, Indomethacin was kindly supplied by Sharp and Dohme.

RESULTS

AND

myometrial obtained

DISCUSSION;

extract by

centrifugation

is

The

distributed at

MntZ in

105,

000

dependent both

soluble

x g for

599

1 h.

guanylate and (Table

cyclase

particulate I).

of fractions

35

‘$ of

the

the

OE

Vol.

89,

No.

2, 1979

Table

BIOCHEMICAL

I : Apparent and

AND

distribution particulate

BIOPHYSICAL

- triton ___--_ pmol/min/mg extract

105.OOOg

34+

supernatant

105.000

- triton _---__ pmol/min/g

231i15

2310+166

4

42i5

46*4

soluble

t triton --__----tissue 15607i1167

87Oi

43oi

in

formed

t triton ---_-protein

25i3

g particulate

COMMUNICATIONS

of guanylate cyclase activities fractionsof rat myometrium.

cGMP _------_-_-___~----_----~~--~~~--

Crude

RESEARCH

20

58

146Oi85

1513&106

14147+

1018

2 g of fresh rat myometrium was homogenized and fractionated as described under “Experimental Procedure”. Aliquots (20-30 pg) of each fraction was assayed for guanylate cyclase activity in the absence or presence of 0.1 % triton,with 1 mM MnC12, 1mM GTP, 10 mio at 37” (standard condicGMP/min/g tissue tions). Results are expressed as total activity, pm01 and specific activity, pm01 cGMP/min/mg protein. Values are the means f standard errors of 6 different experiments, each carried in duplicate.

activity In

was

the

detectable

presence

of

increased in

the

high

activity

was

attributable

sing

the

observed

increment

to

activities

of

on

the

(3-fold)

of

increasing

SNP.

In

SNP

effect

high

as

the

as

1 mM.

the

In

50-100

of

fold), It (16)

is

of

the

FM

SNP;

which possible DTT

that may

prevent

the

there

could

be in

MnGTP

the

present

oxidation

600

effect

almost

sustained

With % of

maximal

stimulatory

was

myometrial

that

basal

II).

for

of

free

then

declined with

1 mM in

concentrations as thiol

of

stimulation

enhancement

SNP

case,

effect

values

a marked

5 mM obtained

stimulatory

of DTT,

Increa-

cyclase

(Table

dose-dependent

reached

DTT,

20

the

detergent

guanylate

activity.

than

1 mM

absence

and 1 mM

more

contrast,

a further

particulate

their

extract

cyclase. by

for

and for

not

the

accompanied

soluble

was

1 illustrate

with

reported

cation

by

guanylate

not

whole

(60 % ). Thus

enhanced

particulate

concentration

concentrations presence

sole

the

1 O-fold;

treatment

As

fractions.

of

increased

after

both

particulate

activity

slightly

1 % was

fractions

activity. at

(6-7

systems

to

(3),

Mn+2

soluble

only

activity.

sources

both

was

of

up

figure

occurred

with

other

extracts

crude

effective

Data

pellet

in

cyclase

in

a maximal

the was

guanylate

activity

cyclase

fractions

preferred

Mg-GTP,

65 % in the

and

predominance

different

preparations

SNP

the

in

concentration

in

soluble

Activity

soluble

triton

the

0. 1 % triton,guanylate

7-fold.

activity

with

in

;

emphasized groups

the as

for critically

Vol.

89,

Nlo. 2, 1979

--Table

BIOCHEMICAL

II : Effects of cyclase activities

AND

MnGTP and of soluble

BIOPHYSICAL

MgCTP on and particulate

basal

RESEARCH

and stimulated myometrial

MnGTP 1mM ---------

Addition --_-------------------------

pmol

--Soluble None

COMMUNICATIONS

guanylate fractions.

MgGTP 5mM --_-__---cGMP/min/mg

protein

25+3

SNP

100

UM

6*2

152110

130*9

-Particulate None

44*5

arachidonic arachidonic

acid

100

pM

acid

200

PM

triton

0.1

triton

t arachidonic

200

46i5

80*7

% acid

lOi *15

200

pM

20+3

370

*21

580

i21

45zt-6 578i

Guanylate cyclase activity was assayed in 105000 g soluble and late fractions either in the presence of 1 mM GTP, 1 mM MnC12 GTI?, 5 mM MgC12 with or without the addition of SNP, arachidonic or/and 0.1 % triton as indicated. 10 min incubation at 37”. Results are the means f standard errors of 3 different experiments each in duplicate.

100

lo Figure cyclase

1_ : Dose-dependent activity

of

effect rat

of

particuor 5mM acid shown carried

1000

SNP(pM) SNP

myometrium.

(0) during tissue homogenization, ducted for 10 min at 37”, in described under’kxperimental indicated concentrations.

17

on

the

105,000

g

soluble

guanylate

DTT (1 mM) was absent (0) or present centrifugation and assay which was conthe presence of 1 mM GTP, 1 mM MnC12 as Procedure”with the addition of SNP as the

601

Vol.

89,

No.

BIOCHEMICAL

2, 1979

AND

\.

E” .E E too.

BIOPHYSICAL

RESEARCH

+DTT

. ____ Control -+SNP

\

8

-----____________ --o-------------------------I 0

60

p:0incuhation4°time

(min)

Figure 2 : Effect of preincubation on SNP-induced myometrial guanylate cyclase. 20 vg protein fraction were preincubated at 37” for the times DTT 1 mM was present (0) or absent (0) during for fig. l0 Interrumpted lines, control activities SNP (50 pM) for the assay period

involved

in

and

high

with

groups

the

tory

occur,

effect.

the

of SNP,

37”

of responsiveness

was

as

The

involvement

SNP

was

lating

minantly culate cyclases

also

less was

the

of

to

stimulation

by

total

of

sulfhydryl

groups

by

no

(4)

than was

for was

triton

fact

on

more

extract

preparation

the

prevented

noticeable

accounted

of

inhibited

the

the

DTT,

in

enzyme

the

unpreincubated guanylate

that

I mM

SNP

activation.

particulate

treated

with

SNP,

the

preparation. to SNP stimulation

602

contrast,

activitywhich

activation

stimulatory and

under

stimulated

and of the

as

optimal also

found

activity

soluble

other

alky-

effect

triton-dispersed for

by

a SH

It was

The

pre-

preparation.

The

activity.

soluble

a progressive

cyclase

fraction

stimula-

the

N-ethylmaleimide,

basal

the

in

resulted

the

SNP

thiol

2. Preincubation

By

% over

unresponsive

figure

an

latter

on

of SNP.

as in

the

DTT

as of

of DTT

the

resulted

50

soluble

by in

addition

SNP

of of

exerted

illustrated

DTT

absence

unmasking

absence

1 mM

the

oxidation

complete

further

the

In

additional

action

is

in

presence

completely

was

the

stimulation of soluble 105.000 g supernatant indicated on the abcissa. preincubation and assay ; solid lines, addition

of

activity.

to a subsequent

indicated

agent,

conditions when

the

sensitive

of SNP

in

cyclase at

in

enzyme

anti-oxidant

guanylate

incubation

full

resulting

preparation,

loss

of

A protective

myometrial of

expression concentrations

may

COMMUNICATIONS

that

predoparti-

guanylate preparation

by

SNP.

Vol.

89, No.

2, 1979

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

/*A A 0 0 Ii

,/I

.

I 50

Loncenrration

I 150

100

200

(PM)

Figure 3 : Dose-dependent sitmulation of particulate guanylate cyclase activity by various fatty acids and hydroperoxides. Guanylate cyclase in the 105,000 g particlllate fraction was assayed in the presence of 1 mM 10 min at 37” as described under “Experimental ProceGTP, 1mM MnC12, dure”, with the addition of the indicated concentrations (A) of arachidonic (AA), oleic, linoleic (lino), y-linolenic (y-lino) acids and (B) of hydroperoxy-arachidonic (AA -GGH), linoleic (lino-OOH) and y-linolenic(y -linoOOH) acids.

The

cation

requirement

SNP-activated cation the

enzyme,

(Table

II).

myometrial

entrations

The

data

fatty

below

maximal

so

extract

of unsaturated table

for

20 pM response

was

attained

particulate

guanylate

different responses

attained

magnitude

as

concentrations,

for of their

compared is

was

clear

that,

fatty

The

PM

603

(for

The

acids.

and

did

concentrations

not

undetecacid, acid

conc-

hydroperoxystimulated pattern appeared

Even

similar

of

of a series

fatty

also

but

sole

y-linolenic the

acids

the

activity

dose-dependent

were

low

as

Mnt2

presence

Increasing

fatty

acids at

100

linoleic

hydroperoxides

to the

as

almost,

hydroperoxides

corresponding the

the

of activation. and

various

with

particulate

in

PM).

3B).

changed

dose-dependent,

loss

(figure the

the

at

50-80

y -1inolenic

with

it

that

a maximum

cyclase

that

show

effective

increased

a progressive

similar

from

as

3A

at

cyclase

became

Activation

reaching

of arachidonic,

Mgt2

markedly

and

in

guanylate

of figure

acids.

resulted

was

that

was

derivatives

activation

myometrial

though or

decline (20-100

of strikingly maximal

even

higher

with

increasing

pM)

the

in

fatty

Vol.

89,

No.

acids

2, 1979

were

consistently

observation buted

their

bation.

The

inhibits

both

reciably stimulate

tend

of

hydroxyl

the

activity

with

donic

acid

t

tions

were

made

that

activation by

patterns

the by

be

in

observed

achieved

with than

activation high

triton

in

the

detergent, vation and

that

Guanylate

in

acids

guanylate

arachidonic

cation,

resulted

cyclase

(fig.

also

decline

in

was

able

the

soluble

of

was

no

and

two

altercyclase

/mg

protein,

with

arachiobserva-

providing

evidence of

occur

via

a direct

of the

the

rat

myo-

a mecha-

4).

(or

particulate in

two

triton

fraction

604

of

the

pattern no

acid.

It was

quite

in

acid

could

was

ultimately

of

more

fatty

be

clear

still

as

evidenced that

only

was

presence

slightly

including

the

of both

effectively effector

acid

between

important the

either was

compounds.

interactions

More

Mg+2

reflect

t hydroperoxyarachidonic

could

by

activation

activity

activity

activated

of

guanylate

actually

classes

Greater

hydrophobic

induced

the

may

modified

to utilize

interaction

arachidonic

cyclase.

when

to activation of

complex

guanylate

cyclase, acid,

contrast

and

known

presence

guanylate

not

from

hydroperoxy

Triton

in

appto

Similar

difference

of

of arachidonic

assay fatty

the

there

hydroperoxides

acid

alone.

concentrations

The

triton

not

a well

preparation

regions

their

t arachidonic

lo)

protein).

result

character

of

the

cGMP

did

which

cyclaseactiva-

cGMP

particulate

might

or

did

PM),

in

pmol

pmol

incu-

radicals.

units.

presence

as

the

hydroxyl

acid

(15

acid,

in

and

arachidonic

inasmuch

2120

attri-

hydroperoxides

incubation

1900

be

mM)

(17)

the

(6,

10 min.

hydrophobic

acids

(l-5

DTT

guanylate

BHA

cGMP/mg

activation

that

or

hydroperoxides

hydrophobic

either

and

pmol

of

cyclase

Finally,

acid:

regulatory

triton

shown).

thiourea

their

with

the

with

(not

not

guanylate

to prevent

thiourea:

generation

fatty

Stimulations

acid)

and

probably,

in

failed

cyclase

acids

associated

differences

acids

15 hydroperoxyarachidonic

guanylate

between

fact

could

indomethacin

arachidonic

1600

effects

This

reactions

fatty

(during

5 mM

components or

the

COMMUNICATIONS

hydroperoxides.

acid

by

(18)

BHA:

the

lipid

with

t

fatty

Most

cyclase

acid

with

involving

these

pM

of

fatty

Furthermore,

responses

300

5 mM

their

during

the

via

RESEARCH

cyclooxygenase

either

acid

acid

arachidonic

metrium

of

scavengers

fatty

was

nism

ability

arachidonic

radical in

and

cyclooxygenase

by

the

strengthened

cyclase.

the

than

that

lipoxygenase the

induced

with

was

guanylate

inhibitor

effective

BIOPHYSICAL

to hydroperoxides

conclusion the

AND

to imply

conversion

affect

ation

more

would

to

tion

BIOCHEMICAL

as alone

Mnt2 (Table

stimulated

the obsertriton as II). by

sole

Vol.

89,

No.

2, 1979

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

..I.’

600-

,::

..::

+tritron

I 100

r 0

ConcentratioZRfpM)

Figure of arachidonic acid and hydroperoxy arachidonic acid - 4 : Effect alone and in combination with triton to activate particulate myometrial puanvlate cvclase. Assay was conducted as described for fig. 3 in the absence (solid lines) or presence (dotted lines) of 0.1 % triton, with the addition of the indicated concentrations of AA and AA-OOH.

fatty

acids

50-75

%,

or

hydroperoxyderivatives

similar

to the

In rat

which

the

sensitive

involved

in

particulate

i. e.

generation and

levels

in

arachidosnic

be

linoleic intact acid,

on

operate

via

than

presumably

cGMP

levels

further

It

is

interesting

pM)were

strips

(data

totaly not

cyclooxygenase

shown).

metabolism

60.5

which

(1). as

was

might

On

be

of

other

ineffective Under pathway,

hand,

the

physiologifatty

peroxide

to note

the

of hydrophobic

a more that

to

the

a result

utilize

involvement

fraction

contributed

demonstrated the

in

cyclase

soluble

those

and

activated,

without

(10-3000

as

enhanced

fully

radicals.

the

(4),

be markedly

It was

The

such

myometrial

MgGTP.

myometrial

more

presence

guanylate

modulated.

pathway

when

acids

no

the

particulate

mechanisms

could

of hydroxyl

was

demonstrate

and

independently

oxidative

SNP

results

soluble

fractions,

mechanism

oleic

of both

activity

substrate

foregoing

activating of

Both

activating or

NO

effect

interactions. cal

to

the

stimulatjory the

could

activation

effect.

the

extract

activities most

triton

conclusion,

myometrial

_ Maximal

that

acid

derivatives arachidonic,

in

raising

cGMP

the

same

conditions,

has

been

shown

Vol.

89,

No.

2, 1979

to induce

elevations

that

acids,

fatty

the

guanylate

activity of

BIOCHEMICAL

of added

established

features levels,

in if

the

deserves

whether

fatty

with the

latter

locally

acid

It is

could

fatty

modulation

of

to not

acids

(e.g. It also

guanylate of

speculate easily

guanylate

investigation.

activation

COMMUNICATIONS

possible

tissue

liberated further

RESEARCH

medium,

Whether

physiological does

(11).

incubation sites.

by

BIOPHYSICAL

content

tissue

activity)

common indeed

CAMP

subcellular

be modulated

phospholipase

to be

in

cyclase

could

tissue

AND

rat

cyclase myometrium

reach

cyclase as still

a result remain may

share cGMP

occur.

ACKNOWLEDGEMENTS: This work was supported by grants from the CNRS, DGRST, INSERM (C. L. 77. 4. 003. 4) and a contribution from the Fondation pour la Recherche Medicale Francaise. We are grateful to Mrs J. Robichon and G. Thomas for excellent technical assistance and to Mrs J. Jalicot for typing the manuscript. REFERENCES: Leiber, D. , Vesin, M. F. and Harbon,S. (1978) F. E. B. S. Letters, 86, 1. 183-187. Diamond, J. (1977) The Biochemistry of Smooth Muscles, N. L. Stephens 2. Eds., Univ. Park Press, 343-360. Goldberg,N. D. and Haddox, M. K. (1977) Ann. Rev. Biochem., g,823-896. 3. 4. Arnold,N. P. , Mittal, C. K., Katsuki, S. and Murad, F. (1977) Proc. Nat. Acad. Sci. , 74, 3203-3207. 5. Craven, P. A. and DeRubertis, F. R. (1978) J. Biol. Chem. ,253, 8433-8443. F. (1977) Proc. Natl. Acad. Sci. , 2, 4360-4364. 6. Mittal, C. K. and Murad, Wallach, D. and Pastan, I. (1976) J. Biol. Chem. , 251, 5802-5809. 7. 8. Glass, D. B., Frey, W., Carr, W. D. and Goldberg, N. D. (1977) J. Biol. Chem. , 252, 1279-1285. 9. Hidaka, H. and Asano, T. (1977) Proc. Naa Acad Sci. 2, 3657-3661. 10. Graff, G. , Stephenson, J. H., Glass, D. B., Haddox, M. K. and Goldberg, N. D. (1978) J. Biol. Chem., 253, 7662-7676. 11. Vesin, M. F. , Do Khac, L. and Harbon, S. (1977) Mol. Pharmacol. 14, 24-37. 12. Steiner, A. L. , Parker, C. W. and Kipnis, D. M. (1972) J. Biol. Chem. , 247, 1106-1113. 13. Cailla, H. L. , Vannier, C. J. and Delaage, M. A. (1976) Anal. Biochem. , 70, 195-202. 14. Lowry, 0. H. , Rosebrough, N. J. , Farg,A. L. and Randall, R. J. (1951) J. Biol. Chem., 193, 265-275. Hamberg, M. and Samuelson, B. (1967) J. Biol. Chem. , 252, 5329-5335. 15. 16. Bohme, E., Graf, H. and Schultz, G. (1978) in Advance in Cyclic Nucleotide Research, 2, 131-143,Raven Press,N. Y. Land, W. , Lee, R. and Smith, W. (1971) Ann. N. Y. Acad Sci. ,180, 17. 107-122. 18. Vane, J. R. (1971)NatureNew Biol. ,231, 232-235. Halliwell, B. (1978) F. E. B.S. Letters, 92, 321-326. 19.

606