L-Arginine stimulates an endogenous ADP-ribosyltransferase

Vol.

178,

August

No. 15,

BIOCHEMICAL

3, 1991

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

1991

L-ARGININE

STIMULATES

Stefanie

Faculty

Received

AND

June

AN ENDOGENOUS ADP-RIBOSYLTBANSFERASE

Dimmeler

of Biology, University of 3,

848-855

and Bernhard

Department Konstanz,

BriineX

of Biological 7500 Konstanz,

Chemistry, F.R.G.

1991

Summary: An ubiquitous biochemical pathway known to synthesize nitric oxide (NO) from L-arginine has been identified in many cell types. Recent studies indicate that besides activating soluble guanylate cyclase NO is likely to have effects unrelated to the known signal transduction pathway. Activation of the soluble NO synthase stimulates an endogenous ADP-ribosylation of a predominant 39 kDa protein, known to be activated by NO releasing agents. This is demonstrated using the cytosolic fraction of rat cerebellum and HL-60 cells. The ADP-ribosylation is suppressed by the known NO synthase inhibitors N-nitro-L-arginine and N-methylL-arginine. These observations indicate that NO derived from its physiological precursor L-arginine activates an endogenous ADPribosyltransferase. B 1991 Academic Press, Inc.

The synthesis nitrogen to

least

inducible lived

of

atom

be at

of

two

is

producing

NO.

oxide

seems

pathway

that

macrophages

(7),

endothelium

(1,ll).

guanylate

to

an increase

in

has

been (8),

Initially

(12)

cyclase

of

vascular

messenger

should

appear

NO

the

an for

the

known

smooth

an

short-

ubiquitous example

cells as

is

of

be

to

and

in

(9,lO)

and

endotheliumto

activate

muscle,

cGMP and thereby

leading

regulating

be addressed.

used: tetrahydrobiopterin;

0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

There

Formation

cerebellar

(EDRF)

second

(l-3).

guanidino

a constitutive

characterized

the

terminal

identified

factor

r To whom correspondence Abbreviations H4biopterin, dithiothreitol.

NO synthase,

neutrophils

the

the

accepted

(4-6)

biochemical

soluble

widely

of

nitric

relaxing

(NO) from

forms

radical

derived

oxide

L-arginine

isoenzyme free

nitric

SNP,

848

sodium

nitroprusside;

DTT,

Vol.

178, No. 3, 1991

the

vascular

tone

Besides

being

directly

activating

involved

system

in

soluble

guanylate

mimicked

cyclase.

by cGMP itself

hypothesis

that

the

or

NO also action

cerebellum

traced

to

of

the

lipophilic

cGMP derivatives, effects

in

in

the

NO and

NO-

activation

most biological

its

pathway

(20).

Therefore,

NO mediates

be

activated

found

action

back

by

seems to of

NO synthase

the

(14).

homeostasis

as a signalling

systems

is

function

wall

cyclase,

as in the

most

platelet

vessel

a role

with

as well

RESEARCH COMMUNICATIONS

cytostatic

play

vasodilators

generating

of

and

(18,19)

forebrain

as

guanylate

and to

Mechanistically

the

mediator

killing

(16,17)

vertebrate

as well

soluble

the

nervous

AND BIOPHYSICAL

(11,13-15)

a potent

in

macrophages the

BIOCHEMICAL

of

actions

are

supporting

by cGMP as the

second

messenger. Interestingly, that

several

NO has

cyclase. cell

It

additional has

been

proliferation

BALB/c

3T3

also

hepatocyte

protein

showed that

ribosylation

of

this

we proposed

ADP-ribosylation

Here synthase rat

reaction

we

like

stimulates

cerebellum

Therefore,

the

to the cytosol

in

ADP-ribosylation

agents

lack

849

cyclic soluble

NO inhibits

cause

tissues

still

way

the

ADP-

(24,25).

In

action

physiological

of

NO

significance

unknown.

of

a distinct

by

an

active

a 39 kDa protein

DMSO differentiated of

a

in

cGMP independent

NO generated

ADP-ribosylation and

cell

several

the is

that

in

cGMP independent

although

inhibit

calcium

that

a partially

a direct,

show

these

NO liberating

presently

free

indications

a 39 kDa protein

on an enzymatic of the

in

guanylate

compounds

achieved

is

concept

of

cytosolic

This

also

the

activation

because

are

synthesis

Own results

respect

decrease

fashion

There

support

NO containing

(21,22).

independent cyclase.

findings besides

shown that

and

guanylate

(23).

effects,

fibroblasts

nucleotide

recent

HL-60 protein

NO using

cells.

may occur

Vol.

178, No. 3, 1991

under

physiological

endogenous

BIOCHEMICAL

AND BIOPHYSICAL

conditions

by

released

from

various

sources.

Materials Preparation

NO

RESEARCH COMMUNICATIONS

of

a cytosolic

and Methods

fraction

from rat

cerebellum

was taken out and A female rat was killed using CO . The cerebellum 0,l mM EDTA, 10 mM washed twice in washing buffer i!0,32 M sucrose, HEPES, pH=7,4). All following procedures were carried out at 4 OC. The capillaries were carefully removed to avoid contaminations with erythrocytes. After washing the cerebellum once more in homogenizing buffer (10 mM HEPES, pH=7,4, 1 mM DTT) the tissue was homogenized and centrifuged (13.000 x g, 10 min). The supernatant was centrifuged at 160.000 x g for 30 min in an ultracentrifuge to Protein was determined using the obtain the cytosolic fraction. Bradford assay. Preparation

of

the

cytosolic

fraction

from

HL-60

cells

HL-60 celis were cultured in RPM1 1640 medium containing 1 mM 2 mM L-glutamine, 100 units/ml penicillin, 100 ).Jg/ml pyruvate, 1% non-essential amino acids and 10 % fetal calf streptomycin, serum at 5 % CO*. To induce differentiation along the neutrophil HL-60 cells (3 x 10 cells/ml) were cultured for 4 days pathway, in the presence of 1,25 % DMSO. The cells were pelleted at 200 x g for 5 min and washed twice with 10 mM HEPES, pH=7,4. Then the pellet was resuspended in buffer containing 10 mM HEPES, pH=7,4 with 5 I.LM pepstain A, 5 /.&M leupeptin, 100 I.~M PMSF and 10 PM sonicated. The cytosolic trypsin inhibitor and fraction was obtained as described for the peparation of the cerebellum cytoso1.

ADP-ribosylation was mainly carried out as previously described by BrUne et al (25). The cytosol (80 kg protein/assay) was incubated in 100 mM HEPES, pH=7,4, 2 mM DTT, 1 j.&M cold NAD and 0,5 PCi [32P]NAD/assay in a total volume of 80 ~1. After an incubation for 90 min (cerebellum cytosol) or 45 min (HL-60 cytosol) at 37 OC the acid insoluble proteins were precipitated with 20 % TCA and centrifuged for 10 min at 13.000 x g. The resulting pellets were washed twice with cold water saturated ether and were resolved in a 10 % sodium dodecyl sulfate-polyacrylamide gel (26). Gels were dried and exposed to KODAK X-ray films at -80 OC. The autoradiography was quantified densitometrically. Thiol solutions as well as solutions of N-nitro-L-arginine, sodium nitroprusside and NADPH were made fresh every day and were pH-balanced before use. Materials

[32P]NAD (800 jKi/mmol) was purchased from DuPont de Nemours, [14C]Methylated rainbow protein molecular weight markers 200 kDa) were bought from Amersham Corp. (6R)-5,6,7,8-TetrahydroL-biopterin was obtained from Dr. Schircks Laboratories Switzerland). All other chemicals were delivered by Sigma, Culture media and supplements were purchased from Laboratories (Grand Island, NY, USA) and fetal calf serum Biochrom KG (Berlin, FRG). 850

FRG. (14.3-

(Jona, F.R.G. Gibco from

Vol.

178, No. 3, 1991

BIOCHEMICAL

Results The

soluble

clearly

and

250

centrations

dependent

(27).

NO-synthase.

the

griess

Or5

neously

we

or

the

We succeeded

as the

ADP-ribosylation

cerebellum

concerning

the

synthase 39 kDa

incubation

like

(fig.l/lane

1 (lane

syltransferase

of

added

activity

NO-production

liberated like

for

ADP-ribosylation

the

the

activation

of

the

nitro-

activates

NO is

in

sponta-

SNP (sodium

if

an

the

en-

question

released

from of

the

cytosolic

NO syn-

NO synthase

as well

cytosolic

E) we detected

all

B).

fraction

of

did

not An

a low basal

39 kDa protein in

increase

in

(fig.l/lane activity

cofactors,

Parallel

the

cofactors

order

rat

active

ADP-ribosylation 851

using

to

cere-

stimulate

labelling

C).

We observed

compared

to

control

densitometrically.

or

5 mM N-methyl-L-arginine

NO of

NO synthase

revealed

that

SNP stimulated synthase the

the a 4-

specific

the

NO

of

by

experiments

suppress

activity

the

quantified

1 mM N-nitro-L-arginine

causing

with

from

occurs

was inhibited

activity. in

NO,

activating

ADP-ribose

inhibitors

of

address

of

a marked

with

A and

activation

cells.

in

without

in

con-

to

ADP-ribosyltransferase

increased

effective

by

finding

Addition

protein

pg/ml

in

order

also

ADP-ribosylation

results

higher

synthase

In

reaction

figure

cytosol.

the

agents

we looked

in

and HL-60

As shown in

results

be

cofactors

fraction

(3-morpholinosydnonimine)

39 kDa substrate

thase.

hibitors

that

NO containing

source

these

to

shown).

ADP-ribosylation

predominant

This

not

known

calmodulin,lO

cytosolic

measuring

demonstrated

SIN-l

physiological

fold

was assured

is

of PM;

(27)

ADP-ribosyltransferase.

whether

Addition

literature

(data

decomposing

dogenous

bellum

in

reaction

prusside)

cerebellum

the

to

14

This

Previously

rat

PM; NADPH, 200 PM; Ca*+,3

described

the

its

from

H4biopterin,

RESEARCH COMMUNICATIONS

Discussion

and

NO synthase

cofactor

(arginine,

AND BIOPHYSICAL

is

in-

the

NO

ADP-riboeven

39 kDa protein

more com-

Vol.

178,

No.

BIOCHEMICAL

3, 1991

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

A B C D E Fiuure 1. ADP-ribosylation using rat cerebellum cytosol. [32P]ADP-ribosylation was carried out as described in Materials and Methods section with following additions. Lane A: cofactors + 1 mM N-nitro-L-arginine; lane B: cofactors + 5 mM N-methyl-L-arginine; lane C: cofactors; lane D: 100 PM SNP; lane E: control; cofactors: 0.25 mM L-arginine, 0.2 mMNADPH, 0.5 PM H4biopterin, 3 FM Ca*+ and 10 pg/ml calmodulin. The shown autoradiograph is representative for three different preparations and reveals a molecular weight range of about 35 - 45 kD.

pared an

to

NO-liberating

about

2-fold

conditions two

agents increase

(fig.l;

further

dioactively

labelled,

stimulation Since

et

reached

al

(9)

linear

increase

ferase

activity

ADP-ribosylation

D to

E).

molecular

linearity an

using

30 to period

20 min,

ra-

from

the

with

the

human platelet

in

90 min. of

90 rat

slow

be explained.

90 min correlates

were

was increased

NO synthase

after

these

unchanged.

incubation the

only

cytosol

weights

between

30 min could

under

cerebellum

39 kDa protein

activity

observed

the

remained

showed that

up to

In

causes

a

The min. cere-

increase

The following

ADP-ribosyltranscytosol

stimulated

SNP (24). As

using In

the

after

a maximal

up to

the

they

showing

was maximal

of ADP-ribosylation

with

of

SNP (100 PM) which

higher

although

manner

Knowles

bellum

lane

slightly

The ADP-ribosylation time-dependent

of

compare

bands with

like

presented the

these

already

in

cytosolic cells

reported

the

figure fraction

existence by

Schmidt

2 the of of et

same

situation

was

DMSO differentiated an active al 852

(28).

HL-60

NO synthase Again

confirmed cells.

pathway

looking

for

was the

Vol.

178,

No.

3, 1991

BIOCHEMICAL

A

AND

B

BIOPHYSICAL

C

RESEARCH

D

COMMUNICATIONS

E

Figure 2. L-arginine stimulates an endogenous ADP-ribosylation. ADP-ribosylation was determined as described in Materials and Methods section using cytosol of DMSO differentiated HL-60 cells. Lane A: cofactors + N-methyl-L-arginine; lane B: cofactors + Nnitro-L-arginine; lane C: cofactors; lane D: 100 PM SNP; lane E: control. Other details as in Fig. 1.

39 kDa protein (fig.2/lane for

E).

the

clearly (fig.

we detected Addition

increased

ADP-ribosylation

C).

Again

B and A)

the

NO stimulated

the

NO synthase

et

one. al

also

of

Our results

revealed

the

of

cytosol

activity

associated

the

N-nitro-L-arginine have

with

rat

other

1,

caused

predominant

the

Besides

suppressed

to

fig.

required a

protein

and N-methyl-L-arginine for

D) of

differences

to

the

activity

cofactors

to

already

a higher cerebellum. bands

(fig.

NO synthase

inhibit

NO originating

NO liberated

%/lane

point

compared

Em-60

equivalent

inhibitors

inhibitors

Similar

(29).

known

(fig.

NO synthase

tive

and all

ADP-ribosylation.

ADP-ribosylation

ADP-ribosylation

N-nitro-L-arginine

pathway,

clearly

basal

L-arginine

NO synthase,

(fig.2jlane

results

of

cytosolic

a/lane

both

a low

from

SNP causes

39 kDa protein. the

from the

Although

ADP-ribosylation being

the

more effec-

been reported activity

our

in

by Gross HL-60

Furthermore,

the

1) was not

cells radio-

apparent

in

cells. These data

NO synthase shown before,

support

and not activates

our only the

view

that

NO derived

from

artificial

ADP-ribosylation 853

from

NO-donors of

the like

a cytosolic

cytosolic SNP,

as

39 kD

BIOCHEMICAL

Vol. 178, No. 3, 1991

Therefore,

the

endogenous

as an inter-

and

intracellular

protein. cells

AND BIOPHYSICAL

sufficient

signal

to

stimulate

generation

the

protein.

Although

the

sylation

is

unknown

we suggest

as the

only

guanylate

still cyclase

synthase

was

found

cells

with

the

cause

cGMP-independent

of

NO from

messenger

molecule

ADP-ribosylation

of

physiological

to

RESEARCH COMMUNICATIONS

significance that

NO not

intracellular

be associated of

rat

a

may be a a cytosolic

the

only

target.

in

ADP-ribosylation

of

different

ADP-ribohas

An active

cerebellum

cytosolic

soluble

and

protein

NO HL-60

and

NO effects. Acknowledsments

The

work

was supported

by the

Deutsche

Forschungsgemeinschaft

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