External GSSG enhances intracellular glutathione level in isolated cardiac myocytes

Vol. 147, No. 2, 1987 September 15, 1987

EXTERNAL

BIOCHEMICAL

GSSG

AND BIOPHYSICAL

ENHANCES INTRACELLULAR

RESEARCH COMMUNICATIONS Pages 658-665

GLUTATHIONE

LEVEL

IN

ISOLATED CARDIAC MYOCYTES

C.Guarnieri,

Department

A.Fraticelli,

C.Ventura,

of Biochemistry,Centre

University

of Bologna,Via

I.Vaona,

of Research Irnerio

and

R.Budini

on Heart

48,40126

Metabolism,

Bologna,

Italy

Received July 30, 1987

The addition of external GSSG at concentrations in the range 50-500 uM produces in isolated adult rat heart myocytes an increase of GSH level and only a slight increase of GSSG level. external GSH at the above same indicated concenOn the contrary, The pretreattrations did not change the cell glutathione pool. ment of depleted the myocytes of the cells with diethylmaleate glutathione and enhanced the GSSG-induced replenishment effect on increase GSH level. On the contrary, the addition of GSH did not the concentration of cell glutathione. The level of cell GSH in diethylmaleate-treated myocytes was not increased after 30 min of incubation with cysteine, or acetylcysteine. The GSSG induced-stimulation on GSH level was not inhibian inhibitor of glutathione synted by buthionine sulfoximine, thesis. On the contrary, this stimulatory effect was inhibited by N, N-bis(2-chloroethyl)-N-nitrosourea, an inhibitor of glutathione reductase, or partially, by the remotion of glucose from the incubation medium. These results support the idea that the isolated adult rat heart myocytes are able to utilize external GSSG in order to increase glutathione pool, probably the intracellular through the reduction of the imported GSSG to GSH. 0 1987 Academic Press, SuikMARY:

IW.

Several

studies

functions status (2)

in (11,

or

diates

from (3).

functions

have demonstrated cells

Abbreviations:

protection

of

the

destructive

effects

it

a number

has long of

the cells

0 I987

of

of from

reactive

been recognized

important

cellular

plays

multiple

cellular toxic

substances

oxygen that

thiol

interme-

glutathione

processes,

detailed

BSO, buthionine sulfoximine; DEM, diethylmaleate; BCNU, N,N-bis (2-chloroethyll-N-nitrosourea.

0006-291X/87$1.50 Copyright All rights

glutathione

such as the maintenance

the

Although in

that

by Academic Press, Inc. in any form reserved.

of reproduction

658

Vol. 147, No. 2, 1987

information

about

te,

except

BY

using

for

the

liver

to

oxygen

reactive

glutathione

is in the

the heart

disulfide

these

disulfures

liver

(9).

Also

the

organ

which

been

described

present

study

myocytes

with

utilizes

highest

we

diethylmaleate

MATERIALS

the

examined

of

is

evidence reactive

it

the

trypeptide

mixed-

to this,

kidney

glutathione

glutathione

that

intracellular

could

(4);

external of

gluta-

is probable

glutathione

ability

oxygen

intracellular

muscIe

the

that

(7). of

trypeptide.The

appears sensitive

cardiac

removes

the

result

against

level

lung

external pool

and there

in the

circulating

to utilize

intracellular

the

last

particularly

and referring

of

that

is

stores

of external

reserve

This

from

is

trypep-

a lower

while

values,

adequately,

(8)

the

of

higher

be removed

utilization

intracellular

the

synthesis

could

that

contractility

sustain

inhibitor

tissues

protection

of muscle

“de nova”

glutathione

the

control

inadequa-

among the mamnalian

(4).

(5)

in

still

a specific

muscle

metabolites

involved

glutathione

have

muscle

is

ascertained

rate

the cardiac

does not

via

(BSO),

which

RESEARCH COMMUNICATIONS

or kidney.

has been

in heart

because

of

it

and kidney

interesting

the

sulfoximine

turnover

is evident

thione

such as liver

biosynthesis

with

If

of glutathione

tissues

has a different

and

synthesis

the

tide

(6)

AND BIOPHYSICAL

buthionine

glutathione

value

BIOCHEMICAL

the

than

is

following

the

the

the primary

CSH (10). isolated

in order

in

augment

recently

adult

level

it

has

In the cardiac

to replenish its

depletion

(DEM).

AND METHODS

Isolated adult cardiac myocytes were prepared accordingly Capogrossi et al.(ll). The hearts were excised from anaesthetized Sprague-Dawley rats (300 g) and in-mediately perfused via the aorcontaining (rrM): 116 NaCl, 5.4 ta at 37O C with a medium buffer KCl, 26 NaHCO3,l NaH2P04,l MgSO4 and 5.6 D-glucose which had been (buffer equilibrated with a 95% 02- 5% CO2 gas mixture (pH 7.4) Following this wash out, a recirculating perfusion was begun A). 659

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 147, No. 2, 1987

with 50 ml of buffer A with an additional 50 mg collagenase (Type I;Sigma) and CaC12 to give 50 uM. After 15-20 min the hearts were removed and the ventricles were chopped with scissors in buffer A containing collagenase. The resulting suspensions were filtered through nylon gauze and the cells were collected under gravity 6 min. buffer A after The cells were resuspended in 20 ml of containing 0.25 r-&i CaC12 and again sedimented under gravity. The myocy tes were then resuspended in medium A containing 10 nfvl Hepes, 1 n&I CaCl2 and 20 n-M D-glucose (buffer B). The pH 7.4, resulting cell suspensions were 70-80% in the rod form and were maintained upon incubation at 37O.C. After diluting the cell to protein concentration of 2 mg/ml, the cells were depleted of glutathione by incubating the myocytes at 37oC in the presence of 0.1 pi/ml diethylmaleate (DEM) dissolved in dimethylsulphoxide. buffer B After 10 min incubation, the cells were washed twice in and were then incubated with different concentrations of GSH or GSSG. After 30 or 60 min of incubation, the cells were collected by centrifugation at 50 g for 2 min and after washing twice with buffer B at 4’C, the myocytes were denaturated with cold 6% PCA. Following a sonification by a Labsonic sonifier cell disrupter (50 w; 10 set), the acid supernatant was separated by centrifugat ion, neutralized with 2 M K2q-0.3 M N-morpholinopropane sulforic acid and assayed inmediately for GSH + GSSG content. For GSSG measurement, the acid solution contained 50 n-M N-ethylmaleimide in order to trap GSH.The acid extracts were neutralized with 2 M K2C03-0.3 M N-morpholinopropane sulforic acid and assayed immediately for GSH + GSSG content according to the method of Tietze (12). The assay mixture for glutathione determination contained : 50 n-M potassium phosphate buffer, pH 7.4; 1 n-M EDTA; 0.1 n-&l 5’5-dithio bis (2-nitrobenzoic); 0.15 n&i NADPH; 6 units/ml glutathione reductase and an appropriate volume of neutralized sample. After 1 min, the increase in absorbance at 412 min was measured for 3 min using a double beam Perkin Elmer spectrometer mod.559. The proteins present in the perchloric-denaturated material were solubilized with 0.3 M KCH and determined according to Bradford (13) using bovine serum albumin as standard.

RESULTS Figure

1

shows

with

different

was

no

of

the

addition

an elevation

tracellular

cardiac (Figure

the

the

was

of

added

intracellular myocytes 2).

cardiac

On the

of GSH (25;

incubated 50;

100;

for

30 min

500 uM)

there

levels.

On

of GSSG at

above

50 pM,pro-

the

at

concentrations

GSH level.

slightly

of

concentration when above

GSH or GSSG did

incubated

contrary,

The

increased

concentrations

levels were

myocytes

GSH or GSSG intracellular

GSSG also

glutathione The

in

concentrations

change

contrary, duced

that

in

the

control

pretreatment

660

of

external

the

the

in-

oxid

ized

100 uM. not

change

conditions of

the

when I the for

cells

60 min for

IO

Vol.

147,

No.

2, 1987

BIOCHEMICAL

Figure

min

100

500

RESEARCH

COMMUNICATIONS

50

25

q

DEM than

whilst

BIOPHYSICAL

1. Effect of GSH or GSSC addition on intracellular GSHU or GSSC concentration in adult rat cardiomyocytes. After 30 of incubation,the cells were collected by centrifugation and after washing, the content of glutathione was determined as described in Methods.

with

lower

50

25

Control

AND

strongly

the

the

levels

reduced during

control

of

GSSG

were

the the

level following

not

modified

GSH 10

4

0.6

3

0.6

/ 1

/

GSH

which

60 min

of

by

drug.

the

remained incubation, When

the

GSSG

10 6

2

of

/

0.4

I ,‘e--w-

----A

02

&&i-i 0

0

0

Figure

30

60

i

0

in control 2. GSH and GSSG levels cardiomyocytes supplemented with Control; -ADEM-treated -omented cells; -w-GSSG supplemented Values are means + S.E. for four

661

I

J 30

and DEM-pretreated rat GSH or GSSG (5OpM). ccl Is;-+GSH supplecells. experiments.

60

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 147, No. 2, 1987 Table

I. Glutathione with compounds

levels in rat cardiomyocytes correlated with glutathione

Treatment

after treatment synthesis

GSH

GSSG (nmol/mg

Treated Treated Treated Treated Treated Treated Treated

(DEM) + GSSG (50pM) + GSSG + BSO (5nM) + GSSG + BCNU (IOOpM) + GSSG - glucose + Cysteine (In-M) + N-acetyl cysteine (Iti)

prot)

0.45

+ 0.02

0.09

+ 0.01

4.5 3.5 0.96

z 0.5 + 0.8 + 0.03

0.89 0.92

5 z

0.40 0.52 0.48 0.37 0.12 0.11

T 5 T T 2 &

1.44 : 0.05 0.04 0.03

0.03 0.04 0.05 0.03 0.02 0.03

BSO or BCNT were added 10 min before the incubation with GSSG. Values are means + S.E. for four experiments. The cardiomyocytes were incubated with GSSG, Cysteine or N-acetylcysteine for 30 min before glutathione determinations.

DEM-pretreated

myocytes

tracellular

concentration

incubation;

while

in

myocytes

cardiac

the

60 min

of

supplemented

of

GSH did

GSSG levels

enhanced

incubation.

with

not

At

DEM,

level

this

during

increased.

with the

50 uM GSH,

change

slowly

pretreated

50 ~J.M GSSG strongly after

were

of

in-

60 min

of

Differently,

the

incubation

cell

time,the

the

GSH

with

particularly

GSSG level

increased

too. Table

1 shows

effect

on

that cell

in

GSH level

reduced

when

the

slightly

when

glucose

Likewise

the

presence

GSH pool tion

of

stimulated the

did

intracellular

incubation

ccl 1s was

of

the

DEM-pretreated

myocytes

produced

the

by external

were

pre-treated

omitted

from

BSO did

with

not

modify

the

by GSSG incubation.

In

not

depleted

of

glutathione

restore

the

cell

concentration

of

DEM-treated

cells 662

GSSG

BCNU

incubation

of

increasing

50 uM

the

myocytes

acetyl-cysteine The

the

or

was more

mixture.

replenishment

addition, with

the

of incuba-

cysteine

or

by

the

alone

or

GSH level.

GSSG was with

increased 50 MM GSSG

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 147, No. 2, 1987 additional from

BSO.

the

The

presence

incubation

produced

mixture

while

GSSG level,

of

the

a lesser

also

presence

evident

BW

or

the

caused of

remotion

an increase

cysteine

enhancing

of

glucose

of

the

cell

or N-acetylcysteine

effect.

DISCUSSION The

present

myocytes

study

to

indicates

external

particularly

when

depleted

by DElvI preincubation.

synthesis

inhibitor

(41, it

mediated utilization

This

of

conclusion

obtained

by

myocytes.

in

adding

part

external

by

the

enyme

glutathione

of

the

cells

with

tathione

reductase

was

mited

recovery

suggests

a probably

glucose lated dent

in GSH

deprivation. ef :fect to

the

on the energetic

GSSG, derives

from

the

not

further

replenishement

undergo

the

the 663

is

1 ikely

reduction

enzyme

effect this

experiments

medium.

In

glutathione

myocytes.

GSH

hypothein

fact,

which the

li-

cells

NADPH that

glu-

on

glutathione-depleted of

the

pretreatment

the

confirm

is of

the of

to

possibility,

replenishment

that

increasing

seems

the

of

and

inhibitor the

ef feet

GSH-depleted

it

production

of

to

fact

from

in

inadequate

level

is

same negative

synthesis,

incubation

level

Another

and

myocytes

reduces

(14),

removed

nova”

an effective

evidence

glutathione

the

reductase.The

Additional

this

with

an effect

acetylcysteine

cardiac

sis. glucose

or

by “de

by external

is

glutathione

such

by the

pool

stimulated

level,

glutathione

interfere

that

confirmed

level

GSH

precursors.

GSSG favour

BGNU,

the

of

cardiac

adult

a known

to

likely

that

GSSG enter

of

BSO,

external

cysteine

Excluding

of

pool

able

very of

myocytes-glutathione that

not

aminoacids

is

cellular Since

appears

the

exposure

an increase

the

is

breakdown

by

the

GSSG produces

evident

replenishement,

that

secondary

the pool

to

GSSG-stimuis

depen-

Vol. 147, No. 2, 1987

The

fact

BIOCHEMICAL

that

glutathione

external

pool

damaged

AND BIOPHYSICAL

GSH does

suggests

that

and show a rather

not

the

RESEARCH COMMUNICATIONS

increase

sarcolenmal

specific

property

intracellular

the

membranes are of being

not

impermeable

to GSH. The ability also

of

evident

external

in control

effect

of GSH level

during

the

(data

not

cells,

shown).

cells,

is

following

in which

level

GSSG to elevate

This

up to 60 min of

The remotion

the

addition

latter

to glutathione

value

GSH level

is

DEM. The maximum while

incubation,

to

decrease

to

the

of GSSG enhances

slowly

DEM-treated

linearly

the GSH

perfusion. external

being

GSSG by cardiomyocytes

condition,such

cells a process

supports could

idea

the

be alternative

synthesis.

the concentration

of external

level

is close

present

to

to suggest unless

tends

is opposite

Since

scle,

with

30 min of

in glutathione-depleted

in this

tempted

treated

after

behaviour

mechanism of

more accentuated that

reached

30 min,

the

not

the myocytes

those this

GSSG could

GSSG able

to

increase

in the circulation

mechanism also pass through

(151,

operates the

the GSH

in the

vascular

we

are

heart

mu-

wall.

ACKNOWLEDGEMENTS The authors thank Mrs. A. Zarri help. This research received financial blica Istruzione (Roma).

for

her

support

invaluable from

secretarial

the Minister0

Pub-

REFERENCES 1. Larsson,

A., Orrenius, S., Holmgren, A. and Mannerwik, B. (1983) Functions of Glutathione, Raven Press,New York. 2. Gilbert, H.F. (1984) in Methods in Enzymology (Weld, F. and Moldave, K., eds.), vol. 107, pp. 330-351, Academic Press, New York. 3. Meister, A. (1983) Science 220, 472-477. 4. Griffith, O.W. and Meister, A. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 5606-5610. teds.)

664

Vol. 147, No. 2, 1987

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

5. Guarnieri, C., Flamigni, F. and Caldarera, C.M. (1980) 3. Mol. Cell. Cardiol. 12, 797-808. 6. Doroshow, J.H., Locker, C.Y. and Myers, C.E. (1981) J. Clin. Invest. 65, 128-134. 7. Talesmik, J. and Tsoporis, J. (1984) J. Mall. Cell. Cardiol. 16, 573-576. 8. Guarnieri, C., Vaona, I., Scheda, M. and Caldarera, C.M. (1986) Free Rad. Res. Comns. 2, 101-105. 9. Harrisch, G. and Mahmoud, M.F. (1980) Hoppe-Seyler’s 2. Physiol. Chem. 361, 1859-1862. 10. Berggren, M., Dawson, J. and Moldeus, P. (1984) FEBS Lett. 176, 189-192. Il. Capogrossi, M., Kort, A.A., Spurgeon, H.A. and Lakatta, E.G. (1986) J. Gen. Physiol. 88, 589-613. 12. Tietze, F. (1969) Anal. Biochem. 27, 502-522. 13. Bradford, M.M. (1976) Anal. Biochem. 72, 246-254. 14. Babson, J.R., Abell, N.S. and Reed, D.J. (1981) Biochem. Pharmacol. 30, 2299-2304. 15. Griffith, O.W. and Meister, A. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 5606-5610.

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