Response of mitochondrial ATPase activity to uncouplers in isolated organelles and whole cells of Zajdela hepatoma

Vol. 100, No. 3,198l June

8iOCHEMiCAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages 1202-1208

16, 1981

RESPONSE OF MITOCHONDRIAL ISOLATED

LuciakGva

Research

March

TO UNCOUPLERS IN

and Stefan

Institute,

Slovak

Bratislava, Received

ACTIVITY

ORGANELLES AND WHOLE CELLS OF ZAJDELA HEPATOMA Katarina

Cancer

ATPase

Kuzela

Academy

of

Sciences,

Czechoslovakia

6,198l

SUMMARY. ATPase activity of coupled Zajdela hepatoma mitochondria was rendered uncoupler-sensitive by decreasing free fatty acids content in mitochondria or by preincubation of mitochondria with ATP prior to the addition of an uncoupler. The latter treatment resulted in an accelerated transport of ATP into the organelles. The effect of carbonylcyanide-m-chlorophenylhydrazone and oligomycin on the decrease of the AW content in whole Zajdela hepatoma cells indicated that the hepatoma mitochondrial ATPase is stimulated by uncouplers in vivo. The conclusion is that the uncoupler-insensitive ATPase zti%?y of coupled Zajdela hepatoma mitochondria is exhibited only by isolated organelles and results from a reduced ATP/ADP translocase activity. INTRODUCTION. mitochondria tumors

/for

The ATPase

/l/

similarly

rev.

see 2/

uncoupler-sensitivity with

ATP prior

effect

of

found

in

ATP is brain

explanation study hepatoma stimulated translocase.

is

the not

that

the

mitochondria ATPase

restored

if of

is

activity

Moreover,

several

tumor

still

the

@I 1981 by Academic Press, of reproducfion in any form

/2-4/.

/4/.

of

ATP to

eliciting

in

an activation

an evidence

is

presented

1202

but

is

also

Satisfactory

resides

Inc. reserved.

This

The results

addition

uncoupler

0006-291X/81/111202-07$01.00/0

incubated

mitochondria

missing.

of the

experimental

are

uncoupler

ABBREVIATIONS: BSA, bovine serum albumin; phenol; CCCP, carbonylcyanide-z-chlorophenylhydrazone;

Copyrighl All rights

other

hepatoma

However,

mitochondria

effect

before

Zajdela

mitochondria

the

to

liver

effect

of

coupled

uncoupler-insensitive.

restricted

this

of

that

addition

and fetal

of

indicate

to

is to

activity

of

this

Zajdela

uncouplerof ATP/ADP that

the

DNP, 2,4-dinitro-

Vol. 100, No. 3,198l

BIOCHEMICAL

mitochondrial

ATPase

is

by uncouplers0

stimulated

AND

activity

BIOPHYSICAL

in

whole

RESEARCH

cells

of

COMMUNICATIONS

Zajdela

hepatoma

?'ATLRIAI,S AWD L1ET-HQDS. rlaintenance and propagation of experimental tumors in ascitic form as IJell as the isolation of mitochondria from these tumors and from rat liver :.Tere performed as described elsewhere /5/. ATPase activity of mitochondria was measured by determination of inorganic phosphate liberated from ATP /6/ in a medium containing 5Om'F'IKCl, 1OmM TRIS-HCl, pH 7.5 and 6rnM ATP. Forward exchange of,ATP by mitochondria was measured essentially according to /7/. ATP content was de-termined in the neutralized RClO& extracts /8/ using commercial assay kit /Boehringer, Germany/. Incubation of mitochondria with carnitine and CoA was performed according to /o/. After the incubation the mitochondria were pelleted and resuspended in the preparation medium and assayed for the ATPase activity and free fatty acids content. Free fatty acids were extracted and determined as detailed in /lo/. RESULTS AND DISCUSSION. Zajdela

hepatoma

concentrations

the

mitochondria of

respectively/

normalized observed

if

uncoupler

of

/Fig.lA/.

It

uncoupler-stimulated be due

to the

a direct

proof

in

the

Zajdela

tumor

that

ATPase to

the

and Ekrlich

ascites

-stimulated

ATPase

of

in

to uncouplers

mitochondria

of

translocase

/12/

than

rat

of

which

by the

ATPase

Dixon

exhibit

activity

1203

role hepatoma

plot it

may

ATP hydrolysis

The rate-limiting

Using

low

However,

FIATPase

mitochondria

ATP hydrolysis

the

was not

ATP/ADP translocase'/l3/

/l/.

the

that

activity.

less

was

mitochondria

factor

contain

a

to

/ll/

tumor

after

/3/

prior

suggested

may be the

questionable.

inhibition

activity

of

CCCP,

mitochondria findings

rate-limiting

activity

by low

previous

activity

carcinoma

ATP/ADP translocase more

the

mitochondria

coupled

only

to the

ATP/ADP translocase

mitochondria

amount

becomes

our

was already

reduced

hepatoma

atractyloside

the

ATPase

an elevated

thus

with

ATP was added

of

to 15).1MDNP or O.l@Y

/up

ATP was added

In accord response

activity

was stimulated

uncouplers

providing

uncoupler.

The ATPase

of

given. and liver uncouplerof

mitochondria carboxy-

was demonstrated

Vol. 100, No. 3,198l

8lOCHEMlCAL

AND

8lOPHYSKAL

RESEARCH

COMMUNICATIONS

Fig.1. Stimulation of ATPase activity of Zajdela hepatoma mitochondria by DNP under various conditions. A- ATP /substrate, 6mM/ was added to the mitochondria 3 min before /o/ or 5 min after /e/ the uncoupler. The preincubation withoATP prior to the addition of the uncoupler was performed at 0 C and that with uncoupler before the addition of ATP at 30°C. B- ATPase activity of Zajdela hepatoma mitochondria prepared in the absence /o/ or in the presence /m/ of 0.1% BSA. ATP was added to the mitochondria after the uncoupler as detailed in A. C- Untreated mitochondria /a/ and organelles preincubated with carnitine and CoA /A/ or preincubated under the same conditions without carnitine and CoA except that the preparation medium was used /A/, were assayed for the ATPase activity. The uncoupler was added to the mitochondria before ATP as detailed in A.

that

the

hepatoma translocase

uncoupler-stimulated and

rat activity

ATPase

liver

mitochondria

activity is

of

limited

both

Zajdela

by ATP/ADP

/Fig.2/.

0

a2 cartloKyatr*ctyloside bvrd I rng protein)

Fi..2. Inhibition of the ATFase activity of rat ZaJde a hepatoma mitochondria by carboxyatractyloside, The inhibition was determined at maximal stimulation ATPase activity by DNP. V is expressed as pmoles of zed /min/mg protein. Rat liver mitochondria in the lOOpI DNP /e/, Zajdela hepatoma mitochondria in the lOp?l DNP /o/.

4-

1204

liver

and Dixon plot. of the ATP hydrolypresence of presence of

8lOCHEMlCAl

Vol. 100, No. $1981

Table

1. Effect

AND

8lOPHYSlCAl

of various free fattv

the

RESEARCH

COMMUNICATIONS

treatments of mitochondria acids content.

;i;;I;toma 1 Free fatty

acids

on

upon treatment

Control values /lOO$/ for rat liver mitochondria 7.3+0.8 nmol/mg protein and for Zajdela hepatoma mitochondriz 18.922.3 nmol/mg protein. Mitochondria were incubated 3 min with 6mM ATP at O°C or with carnitine and CoA. After the incubation mitochondria were pelleted, resuspended in the preparation medium and free fatty acids and ATPase activity were determined. BSA /O.l$/ was included in the preparation medium.

Free

fatty

ATP/ADP

acids

translocase

mitochondria dria

of

with

data

in

tumor

/14/.

liver

/Tab.l/.

demonstrating

the

sensitive

to

However,

as detected

into

Zajdela

The rate depend free

of

to

fatty

order

acids

the

initial

mitochondria

of

inhibit

into the

rat

of

of

ATP/ADP

1205

of

mitochondria acids the

ATPase

activity.

exchange

of

the

ATP

ATP transport higher

uncoupler

mitochondria

when /Tab.2/.

did

ATP and uncoupler.

translocase

fatty

and CoA

3 times

the

ATPase

free

under

to

liver

of

by ATP even

forward

prior

acids

fatty

rate

addition the

fatty

The free

was about

mitochondria

of ATP transport on the

Tab.l/.

uncoupler-stimulated

CCCP, the

the

agreement

content

hepatoma

affected

mitochon-

stimulation

the

Zajdela

by measuring

hepatoma

ATP was added

of

/Fig.lB,C;

the

in

by BSA or carnitine

significantly

eliciting

in

of free

a reduced

of

hepatoma

than is

amount

Decreasing

activity

uncouplers

presence

higher

inhibitors

Zajdela

observation

mitochondria

conditions

the

/ll/.

ATPase

was not

times

exhibiting

in hepatoma

rendered

in

an elevated

mitochondria

content

This

occuring

content

to be 2.6

by uncouplers

acids

naturally Their

was found

rat

activity

in

represent

competetively

not As

BIOCHEMICAL

Vol. 100, No. 3,19Bl

Table

BIOPHYSKAL

RESEARCH

COMMUNICATIONS

2. Rate of the ATP transport into Zajdela hepatoma rat Ilver mitochondria in the presence of CCCP.

Source

liver

Zajdela

and

Initial rate of ATP transporg /nmOl ATP/min/mg protein at 0 C/ ATP added before CCCP ATP added after CCCP /A/ /w

of

mitochondria rat

AND

hepatoma

2.96

2.48

1.86

0.52

/A/ ATP /lmN/ was added to the mitoch Qd ia 10 min before 2w CCCP. After further 5 min incubation! * CTATP /final specific radioactivity 1000 cpm/nmol/ was added. /IV Kitochondr& were preincubated for 5 min with 2p CCCP then ATP /lmM/ and[ C]ATP /final specific radioactivity 1000 cpm/nmo l/ were added simultaneusly. The translocation was l25W carboxyatractyloside 10 min after the addition and mitochondria were separated by rapid centrifugation.

/15/

it

may be supposed

chondria

results

locase

without

A necessary

in

that

a removal

affecting is

of free

concentrations

of

these

total

that

fatty

addition

of

their

corollary

distribution

the

uncoupler

inhibitors amount

such

acids

of ATP to

the

from

in

mitothe

mitochondria.

an intrsmitochondrial can be prevented

added

to

the

trans-

reby higher

mitochondria

before

ATP. Accumulation pase

treatment

a loss

of

et

/17/

al.

isolated

using

the

of mouse

liver

mitochondria

medium data

a milder their

ATPase

possibility

data

whether

activity

the

activity

this

by uncouplers of

activity

was sensitive

ATPase

effect

study of

the to

give

Zajdela in --

CCCP and oligomycin

1206

Kaschnitz

and BSA in

in

by

previously

ATPase

disruption

can be stimulated

/16/.

rnitochondria

presented

the

accompanied

activity

when tumor

cell

or by phospholi-

is

uncoupler-insensitive

and the

question

by ageing

ATPase

that

exhibit

rnitochondria this

acids

reported

to

These

fatty

uncoupler-sensitive

reported

ration

of free

vivo.

were prepa-

uncouplers. raise

to

hepatoma To check on the

ATP

BIOCHEMICAL

Vol. 100, No. 3,198l

Table

3. Effect

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

of oligomycin and CCCP on ATP content Zajdela hepatoma cells,

Additions

ATP content /nmol/ml packed

none

in

cells/

1112.8

oligomycin+CCCP

488.9

CCCP

263.5

Packed cells /0.18 ml corresponding resuspended in 0.82 ml 0.8% NaCl,

to 1Omg protein/ were 0.002% KCl, 0.002% KH PO,+ and 0.0917% K HP0 with or without the inhib&tor/s/. AT? content was determine8 af t er 10 min incubation at 25 C. 5Ofl CCCP and 25pg oligomycin/mg protein were used.

content mined.

in It

content alone the

lwhole

was found upon

presence

chondria

Ehrlich

of were

/W/,

both

which

of

Zajdela

decrease the

cells

than

tha-t

hepatoma

in in

the the

caused

CCCP and ol?gomycin ascites

exhibit

obtained

of

the

pronounced

of

for

cells

that

incubation

was more

results

ascites

/data

/Tab.3/. cells

shown/.

presence

of

/isolated

These

mitoactivity

observations

that

of

Zajdela

hepatoma

is

lost

Kiss

ACKNO*LL,EDGE!,USNTS: We are indebted to Nrs. H. Ferancov6 J. Vagnerova for excellent technical assistance.

preparation

of

in --

the

in

suggest

mitochondria

vivo

CCCP

Similar

ATPase

can stimulate

ATP

by incubation

uncoupler-sensitive not

intracellular

to

during

uncouplers

carcinoma

was deter-

ATPase

and this

allow

activity

capability

mitochondria. and

REFERENCES 1.

Kolarov.

J..

Ku.Zela.

$%.. KremnaskG.

. -. kedersen,

V..

and U
V--

P.L., /I~~$ Prog,.Exp. Tumor --- ~~, -,-, Karger, Base1 K., and Kuzela, !?., /1979/ Neoplasma 26, 691-696 3. &uciakov&, 4. Hayashi, J.-I., Yonekawa, H., Gotoh, O., and Tagashira, Y., /19f30/ Biochem. Biophys. Res. Commun. 92, 261-267 $., Kolarov, J., and Krempask?, V., /1973/ Neoplasma 5* Kuzela, 20, 623-630 2.

1207

Vol. 100, No. 3,15’Bl

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

fSQ Lindberg, O., and Ernster, L., /1956/ Methods Biochem. Anal. 3, l-12 7. Out, T.A., Valeton, E., and Kemp, A., Jr. /1976/ Biochim. Biophys. Acta 440, 697-710 8. Youshok, W.D., /1971/ J. Biol. C&em, 246, 1607-1617 9. Christiansen, E.N., and Davis, E.J., /1978/ Biochim. Biophys. Acta 502, 17-25 10. Bieber, L.L., Petterson, B., and Lindberg, O., /1975/ Eur. J. Biochem. 58, 375-381 R.L., and Ghan, S.H.P., /1978/ J. Biol. Chem. 253, 11. Barbour, 12. 13. 14. 15. 16. 17.

%~~l~75& Kolarov Krempask? Hatalova I., Lakota, J an; Ui&zy V '/:6$7/ NeoplasAa'% 55o-563' Kiiarov, J., Khei:, $i, Krempask?, V.,'Lakota, J., and FEBS Lett. c6, 3?3-3?6 Ujh&zy, V., /1??8/ ?ojtzak, L., and Zaluska, H., /1967/ Biochem. Biophys. Res. Commun. 28, 7691 Morel, F., Laquin, G., Lunardi, J., Duszynski, J., and /lc74/ FEBS Lett. 3?, 133-l% Vignais, P.V., Biochemistry 5, 3887-3903 Chefurka, 'g., /lo66/ Kaschnitz, R.M., Hatefi, Y., and Morris, H.P., /lo76/ Biochim. Biophys. Acta 44o, 224-235

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