The effect of ruthenium red on the uptake and release of Ca2+ by mitochondria

Vol. 50, No. 3,1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMIJNlCATlONS

THE EFFECTOF RUTBENIUM REDONTHE UPTAKEAND RELEASEOF Ca2+ BY ?fITOCHONDRIA by

Carlo S. Rossi, Prank D. Vasington and Ernest0 Carafoli* Institute

of Biological

University

Chemistry

of Padova, Italy and

Institute

of General Pathology

University Received

of Modena, Italy

November 30,1972

summary 2+ Ruthenium red, an inhibitor of Ca binding and transport by mitochondria, by mitochondria only if it is added to the assay promotes the release of Ca2+ mediumbefore the accumulation of Ca2+ has been completed. Once essentially all of the Ca2+ in the mediumis taken up by mitochondria, ruthenium red does not induce its release. It is pro osed that ruthenium red inhibits Ca2+ transport by competing with Ca2+ for Ca2P binding sites, possibly Ca2+ carrier molecules, on or within the inner mitochondrial membrane. In recent studies Vasington et al.

(1,2) confirmed and extended the

observation of Moore (3) that ruthenium red inhibits

They showed that ruthenium red also inhibits

uptake of Ca2+ by mitochondria. the energy-linked

uptake of Sr

and the low- and high-affinity addition, slightly

2+

and ?in'+, the K+-driven translocation 2+ binding of Ca by mitochondria (1,2).

ruthenium red was found to inhibit higher concentration

2+ of Ca In

mitochondrial

than those which inhibit

respiration but at 2+ Ca uptake and binding

studies, Vasington et al. (1,2) also reported that 2+ ruthenium red neither induces the release of Ca from Ca'+-loaded mitochondria 2+ nor affects the dinitrophenol-stimulated release of Ca by mitochondria. (1,2).

In their

the energy-dependent

earlier

In this communication we report that ruthenium red can promote the release 2+ of Ca2+ if it is added to the assay mediumbefore the process of Ca accumu* This work was performed while one of us (FDV) was a visiting scientist from the Biochemistry and Biophysics Section, Biological Sciences Group, University of Connecticut, Storrs, Conn., USA. Copyright 0 1973 by Academic Press, Inc. AN rights of reproduction in any form reserved.

646

Vol. 50; No. 3, 1973

lation

BIOCHEMICAL

by mitochondria

reported

in this

and binding

has been

paper

completed.

we propose

by mitochondria

possibly

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Ca2+ carrier

that

0.25

M sucrose

and were

a combination strip-chart

pore

rapid

measured

(0.45

according:

concentration

the

twice

from

(4).

size).

to Reynafarje

was determined

inhibits

observations

Ca

2+

transport

Ca2+ binding

inner

Wistar

sites,

mitochondrial

strain

The H+ movement

was measured

of aliquots

n pore

of the

membrane.

AND METHODS

isolated

Ca2+ uptake

filtration

membranes

with

and a Beckman Expandomatic

recorder.

following

were washed

electrode

red 2+ for Ca

on or within

MATERIALS mitochondria

ruthenium

by competing

molecules,

Rat liver

On the basis

pH meter

reaction

by a buiret

connected

(5).

in with

to a

previously

medium

(1)

through

milli-

Ca 2+ binding

and low-affinity

and Lehninger

rats

was measured

as described

of the High-

albino

Hitochondrial

was

protein

reaction.

RESULTS Effect

of ruthenium In earlier

red on the release

studies

efflux

of mitochondrial

after

Ca2+ accumulation

find

that

assay

ruthenium

measuring

of Ca2+ (6).

had been

release

caused

State

this

experiment

by the yielding

4 to State

release the

disappearance

of H’ ions

ejection

an H” ejected/Ca2+

of H+ which

succinate

as the

into

the

ratio

847

it

We now

is

added

(Figure of Ca

2+

parallels

1).

in

In the

was followed

of 800 natoms

by

of

substrate respiration

extramitochondrial

the

to the

the movement

respiratory

of mitochondrial

the

system

of mitochondrial

of Ca2+ from

accumulated

test (1,2).

the addition

3 transition

of 750 natoms

of 780 natoms

completed

and release

1A that

not promote

to the

has been completed

and uptake

medium containing

did

of Ca 2+ if

the release

1, the uptake

Ca2+ to an assay

and an immediate

red

by mitochondria

can be seen in Figure

a typical

ruthenium red was added

Ca2+ accumulation

counter It

that

from mitochondria

2+ Ca , ruthenium

shown in Figure the

showed

red can promote

medium before

experiments

which

of calcium

medium.

In

H+ was accompanied

extramitochondrial

good agreement

with

medium, the value

Vol. 50; No. $1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

r 7‘

bl+Yr’ T H+rjoction 2oonAtomso (2SOnAtomsf f 4 f

A

B

Figure 1. Effect of ruthenium red (RR) on the uptake and release of Ca2+ and H+ ions. The reaction mixture contained 80 mMNaCl, 2 mhiTris-NC1 (pH 7.4), 5 mMsodium succinate and 10 mg of mitochondrial protein in a final volume of 3.8 ml. Where indicated 800 natoms of Ca2+ (labelled with 45Ca2+) and 200 nmoles of ruthenium red (20 nmoles per mg mitochondrial protein) were added. Oxygen consumption and II+ movementwere measuredas described in Materials and Methods. The incubation temperature was 25".

of 1 obtained in the absence of a permeant anion (6). Ca2+ uptake, the respiration ceased.

rate

Upon the completion of

returned to State 4 and net tf ejection

The subsequent addition of ruthenium red caused neither

Ca2+ nor an uptake of B+ ions (Figure 1A).

a release of

When ruthenium red was added before

all the Ca2+ in the assay mediumhad been taken up (Figure 1B) there was an immediate reversal

of H+ ejection,

a decrease in respiration

the State 4 rate and the appearance in the extramitochondrial

to approximately mediumof about

90 per cent of the added 45Ca2+ (data for 45Ca2+ uptake not shown). The amount of H+ ions reabsorbed by mitochondria following

the addition

of ruthenium red varied somewhatfrom experiment to experiment, ranging from approximately variability addition

30 to 100 per cent of the amount of H+ ions ejected. may be due primarily

This

to the time period which elapsed between the

of Ca2+ and ruthenium red.

In any case, the uptake of H+ prowted by

the addition of ruthenium red was associated with a stoichiometric release of mitochondrial Ca2* as shown by a final Hf ejected/Ca 2+ accumulated ratio of

848

Vol. 50; No. $1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TARJ.E I

OF RJJTIJE‘JILM RED ON TIiE PELEASE A'JJI UPTAKE OF Ca '+ AND Jl+ IOYS

EFECT

The reaction

mixture

described

in Figure

Ruthenium

red

but

before

final

ruthenium

1, except

that

(RR) was added

all

amount

and conditions

of it

red was determined

reaction

and I!+ ions

after

the

after

were

45Ca2+

as shown in

ejected

final

the same as

protein

mixture

up by mitochondria

of Ca2+ accumulated

were

5 mg of mitochondrial

to the

was taken

of incubation

steady-state

(809 natoms)

Figure

following

used.

1.

The

the addition

levels

were

of

reached.

I!+ ejected

, Ca2+ accumulated

(natoms)

(natoms)

No addition protein) protein) protein)

RR (10 nmoles/mg RR (10 nmoles/mg RR (10 nmoles/mg ---

approximately H' ejected reached

1 (Table

after

the addition

the ruthenium

red

0.95

150 265 190

115 330 180

1.3 0.80 1.05

the effect

complex

or of

a concentration

range

K2RuC16 does not

by mitochondria.

It

inhibit is

of ruthenium

Neither

red exerts

on either

mitochondrial

shown in

Vasington

et al.

-stimulated

Table

amount

state

of

level

compound

II 849

its

range

maximal

K2RuC16 also

of

COW the

release

of 5 - 50 u??, inhibitory

had shown

respiration that

caused

respiration (1.2)

effect

ruthenium

In the

ruthenium

2+

steady

two other

of H+ by mitochondria.

Ca

the

red was a specific

itself,

tested.

2+ of Ca .

uptake

at the final

I,

red.

ruthenium

in which

RuNOCl3 has no effect

shown in Table

was measured

K2RuC16 and RuNOCl 3' were

energy-dependent

experiments

whether

the reabsorption

that

409

of ruthenium

of Ca2+ nor

effects,

380

In the

and Ca2+ accumulated

To determine

pounds,

I).

II+ ejected/ Ca2+ , accumulated

or the earlier

and Ca 2+ uptake has no effect

Vol. 50; No. 3,1973

on the highTable

II,

earlier

BIOCHEMICAL

and low-affinity

ruthenium findings

Despite transport

its

lack

phosphate

inhibit partially

Ca2+ binding sorption

that

the early temporarily

for

that

its

sites

other

that

that

the binding

binding

sites, The inhibitory

specific

property

completely

across

are not

of Ca2+ caused rendering action

Ca

2+

addition

membrane

in this

paper,

such temporary

and a concurrent all

free

the

to ruthenium a conformational

of ruthenium ruthenium

850

red;

change

to ruthenium red

or

exist

taken

of

up suggests

Possibly

the

sites

to

possibility

is

of the temporary red.

on Ca 2+ transport

red molecule

2+

during

the release

binding

another

2+

any Ca

do indeed

red.

temporary

Ca

red,

to induce

Ca2+ in the medium had been

from

red,

in solution

sites

to ruthenium

reab-

membrane

to ruthenium binding

other

of the

of ruthenium

of ruthenium

accessible

we

and possibly

the mitochondrial

them inaccessible

of the entire

overcomes

essentially

accessible

transported available

of respira-

mitochondrial

must have been either

of the

these

the mitochondrial

the Ca 2+ carrier,

upon

no longer

Ca2+ had been

sites

inner

of bound

the inability

that

Inorganic

completely

reported

with

were

appears

inhibition

across

the

was released

If

accumulated

it

4 respiration

(7).

in many experiments

release.

were

State

red

almost

2+ transport

a release

which

Ca2+ once all

these

our

and Ca 2+

mechanisms.

it

within

to sites

the mitochondrion,

accumulated

confirming

respiration

ruthenium

manner Ca

of Hf ejection

bound

accounting within

transported

phase

the

competes

Since

up by mitochondria had been

As shown in

binding,

However,

of the observations red

of II+ ions.

taken

that

causing

sites,

(1.2).

whereas

2+ carrier a Ca

ruthenium

-stimulated

by different

competitive

On the basis that

2+

red

nature,

in

involves

propose

of Ca

2+

K2RuC16 inhibits

overcomes

has been proposed

(5,8-11).

on Ca

respiration

K2RuC16 inhibition

membrane

types

by mitochondria,

an uncompetitive

It

both

as does ruthenium

only

in

inhibits

of effect

and binding

two compounds

of Ca 2+ by mitochondria.

binding

(1.2).

as effectively

tion

red

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

since

appears neither

to be a K,RuC16 L

Vol. 50, No. 3,1973

BIOCHEMICAL

AND BIOF’HYSICAL RESEARCH COMMUNICATIONS

TABLE II

Effect binding

of ruthenium

of calcium

sucrose,

by rat

10 mM Tris-Cl,

at 0'

for

liver

mitochondria.

protein

in

and law-affinity

The system

1 x 10m6 M rotenone,

pH 6.6,

and 5 mg mitochondrial out

red and of K2RuC16 on the high-

a volume

2.5

of 2 ml.

contained:

250 mM

ug Antimycin

Incubation

A,

was carried

two minutes. High-affinity binding nmoles

Ruthenium red (5 nmoles/mg protein)

Low-affinity binding

Ca 2+/mg prot.

Ca2+ added No addition

Inhibition %

nmoles

Ca2+ bound

4.0 8.0

3.5 5.0

4.0 8.0

4.0

Inhibition x

Ca 2+/mg prot.

Ca2+ added

Ca2+ bound

-

80.0 160.0

18.0 22.5

-

1.0

71

1.8

65

80.0 160.0

9.5 15.5

47 33

3.0 55.5

14 0

80.0 160.0

18.5 22.0

0 0

K2Ru$j

(10 nmolesfmg

protein)

8.0

nor

RuNOCl

have

3

mitochondria. transport

Moore

(3)

on Ca showed

by mitochondria

Since the

any effect

high

ruthenium

such

compounds

This

view

is

mitochondrial

binding,

earlier

that

transport,

and release

RuC13

has

also

by 2+

no effect

on Ca

(3).

red has been used

sensitivity

that

2+

of the Ca

2+

transport

may be involved

supported

by the

glycoprotein

that

as a stain

in

recent

for

process the Ca

2-b

isolation

has a high

muco-

to ruthenium

transport

for

red suggests

system

by Sottocasa affinity

and Elycoproteins,

(l-3).

et al.

(12)

Ca*+ (13,14).

ACKNOWLEDGMENTS This (Centro

research per

was supported

lo Studio

della

by the Italian

Fisiologia

dei

851

National

mitocondri

Research and Contract

Council No.

of a

Vol. 50, No. 3,1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

6902109-115.345.0) and by the University The collaboration

of Connecticut Research Foundation.

of Dr. Roberto Tiozzo and the expert technical

of Mrs. Daniela Ceccarelli-Stanzani

arc gratefully

assistance

acknowledged.

REFERENCES Vasington, F. D., Gazzotti, P., Tiozzo, R., and Carafoli, E. Biochim. Biophys. Acta 256, 43 (1972). Vasington, F. D,, Gazzotti, P., Tiozzi, R. and Carafoli, E., in Azzone, 2. E. and Siliprandi, G. F., Carafoli, E., Lehninger, A. L., Qualiariello, N., Eds., Biochemistry and Biophysics of the Mitochondrial Membrane, Academic Press, 1972, p. 215. 3. Pbote, C., Biochem. Biophys. Res. Comm.42, 298 (1971). 4. Schneider, W. C. in W. W. Umbreit, R. Burris and J. E. Stauffer, Eds., Manometric Techniques, Burgess Publishing Company, Minneapolis, 1956, p. 188. Reynafarje, B. and Lehninger, A. L., J. Biol. Chem.244, 584 (1969). 5. 6. Lehninger, A. L., Carafoli, E. and Rossi, C. S., Adv. Enzymol. 29, 259 (1967). Rossi, c., Gazzotti, P., Vasington, F. D., and Carafoli, E. Symposium 7. on Mitochondria, Bari Meetings, Academic Press (In Press). 1973. 8. Mela, L., Arch. Biochem. Biophys. 123, 286 (1968). Chance, B. and Mela, L., Proc. Natl. Acad. Sci. U.S. 55, 1234 (1966). 9. 10. Chance, B., Azzi, A. and L. Mela, in G. Tosteson, Molecular Basis of MembraneFunction, Prentice Hall, NewYork, 1969, p. 561. 11. Lehninger, A. L. and Carafoli, E., Biochemistry of the Phagocytic Process, J. Schultz, ed., North Holland, Amsterdam, 1969, p. 9. 12. Sottocasa, G. L., Sandri, G., Panfili, E., and delernard, B., FEBS Letters l7, 100 (1971). 13. Carafoli, E., Gazzotti, P., Vasington, F. D., Sottocasa, G. L., Sandri, E. and delernard, B., in Azzone, G. F., Carafoli, E., G,, Panfili, Lehninger, A. L., Qualiariello, E. and Siliprandi, N., Eds., Biochemistry and Biophysics of Mitochondrial Membranes, Academic Press, 1972, p. 623. E., delernard, B., Gazzotti, P., 14. Sottocasa, G., Sandri, G., Panfili, Vasington, F. D. and Carafoli, E., Biochem. Biophys. Res. Comm.47, 808 (1972). 1.

852