Unusual effects of a substituted thiophen on mitochondrial swelling, contraction, and oxidative phosphorylation

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 28, No. 6, 1967

UNUSUALEF5ZCISOFASUDSTI'lWlWTHIOPHRNOIVMI!POCHONDRIAL SWELLING, CONTRACTION,AND OXIDATIVJ3PK~PHORYLATION J. B. Peter and L. D. Lee Department of Medicine UCLA School of Medicine Los Angeles, California go024

ReceivedAugust22,

1967

The cytotoxic

immunosuppressive agent,

3-acetyl-5-(4-fluoro-

benzylidene)-2,5-dihydro-4-hydroxy-2oxothiophen, inhibits

succinate

and NAD-linked oxidations

aria but does not inhibit As reported

1967 0).

potent uncoupler tions

(ICI 4~76

oxidation

of ascorbate

herein this

of succinate

substituted IV&linked

=a

less than those which cause respiratory

cx1s studies

show that uncoupling

ion of mitochondria

which subsequently

effects

are noted with rat liver

muscle mitochondria to shw swelling under a variety Rat skeletal centrifugation mechsnical

even though previous

after shaker.

(Peter aa

homogenization The technique

he,

1967).

meter.

Simultane-

of ICI cause contract-

These

and with rat skeletal have failed

muscle mitochondria

1963).

were isolated

by differential

with glass beads in a C02-cooled employed has been described

Oxidative

phosphorylation

at 5X, mp, 26O in a Gilford

Unless otherwise

inhibition.

investigators

at 260 in a Gilson oxygraph with simultaneous and contraction

in concentra-

of respiration.

(Tata & a.,

muscle mitochondria

is also a

oxidations

of rat skeletal

of conditions.

e,t al,,

swell as the ICI concentration

mitochondria

and contraction

mitochon-

(Franklin. thiophen

concentrations

is raised to levels which cause inhibition

detail

by rat liver

or ICI)

was studied

observation recording

in

of swelling

spectrophoto-

stated the oxygraph and epectrophotcxnetric

1053

Vol. 28, No. 6, 1967

studies

BIOCHEMICAL

utilized

identical

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

media containing

and 15 mM DL-malate or 45 mM succinate m,

30 mM pi,

noted in the figures.

are

commercially

available.

in dlmethyl

in concentrations the parameters

plus 0.02 mM rotenone),

and about 0.5 mg mitochondrial

Other additions

dissolved

(15 mM pyruvate 25 mM

8.0 mM MgS04, 0.5 mM EDTA, 50 mM KCI, 0.17 bovine

serum albumin/ml

purity

substrate

protein/ml,

All reagents

The substituted

sulfoxide

(IWO)

thiophen

Immediately

less than 51 (volume/voluw)

before

pH 7.4.

were highest (ICI)

was

use.

had no effect

INSO

on any of

studied.

SUBSTRATE

DEPENDENT

AND INDEPENDENT OF ICI

EFFECTS

Nil0

Nil0

PVRUVATE YALATE

NO SUBSTRATE

in( y pI_ II .02 .I3 .04 .05.55

NO SUBSTRATE

PTWJVATE YALATE

6

I

16

34

42

6

8

30

42

TIMEtMIN)

Figure

1. Standard medium and conditions

as described

In the text were

employed.

F5.nalvolume

in oxygraph was 2.0 ml and In speCtr0

photometer

was 3.0 ml.

Protein

albumin/ml

and 0.83 mg mitochondrlsl

concentration protein/ml.

wa8 0.17 mg

BIOCHEMICAL

Vol. 28, No. 6, 1967

ICI

AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

INDUCED VARIOUS

UNCOUPLING SUBSTRATES

WITH

O 58 mg PROTEIN

STANDARD MEDIUM TOTAL VDLUME=Zml T=26, ,,H=74

Figure

\

2. medium

stand-a

conditions

tdna

a8 described

in text were em-

plopa.

ICI 47776 total

protein

oxidative At this

(ICI)

In average concentration

(mitochondrial

phosphorylation concentration

mitochondria

piration

by the increased

and cause substrate

(Figure

1).

Similar

blphaeic

ICI are noted with auccinate strate

uncoupling

The uncoupling

of respiration

Low concentration8 (Franklin

3).

independent effects

reepiration

1).

of the

absorbance at 520 mb whereas total

protein

swelling

present,

and mitochondrial

inhibit

res-

of the mitochondria

with increasing

and rotenone

concentration

of

but with this

aub-

of ICI (Figure

2)

contraction

induced

of the presence of added Pi, ADP and Mg".

of ICI stimulate

& a&l967

ICI releaee

polee/mg

(Figure

dependent contraction

occurs with lower concentrations

by ICI ia independent

(Figure

.d+5

uncouples

malate as substrate

of ICI a substrate

approaching

01 mM or .02 @oles/mg

plus added albumin)

with pyruvate

is manifest

concentrations

protein

of

Mg++-dependent

a) but our data ehow that in state

This indicates

that

4 despite

low concentrations

of

the presence of oligomycln

the uncoupling

1055

ATPase of mitochondria

is not an AT&se

Vol. 28, No. 6, 1967

effect

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and is not due to hydrolyaie

phosphorylated

high-energy

compound.

FAILURE [02]=

of any hypothetical

OF

OLIGOMYCIN

TO BLOCK

UNCOUPLING

BY ICI

500mpMolcs

2OPQ OLIGOMYCIN

.0025 \

.0050

.0075

.a;00

.0150

.oio I

FINAL

ICI

CONC (mMl [02]= 290 m,u Moles

I 4

I 6

I 12

I I6

I 20

TIME

I 24

I 26

I 32

I 36’

(MIN)

Figure 3. Standard medium and conditions

a6 described

in text were em-

ployed.

Octylguanidine 4) and uncoupling

(0.2 mM) Inhibits

induced by low concentrations

Although octylguanidine

malate. contraction inhibits

with succinate the ICI-induced

that the octylguanidine piration

contraction malate; tiona

contraction

also completely

uncoupling.

inhibits

biguanidine

(DBI) (Prerruman, 1963)

whereas, 0.2 mM DBI has no effect show that octylguanidine

with the idea

In inhibiting

DPN-linked respiration

in the presence of euccinate

mitochondrial

it only partially

This is compatible

Is not as effective

res

(Pressman 1963). inhibits

plus rotenone (Figures

rrucclnate

ICI-induced

or pyruvate plus

5,6).

and WI block the interaction

1056

(Figure

of ICI with pyruvate-

plus rotenone as substrate

as it is In inhibiting

3.0 mM phenethyl

mitochondrial

These obeerva of ICI with

Vol. 28, No. 6, 1967

BIOCHEMICAL

Effect

of

Octylguanidine

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

on

ICI-

Induced

Structural

Changes

1.35(1

1.200

z “,

or itOH 1.050

*

0.900 .33 ******** Succinate -Succinate -Pyruvate ---Pyruvate

0.750

i\

\

(45 mM)+ Rotenone (0.02 mM) (45mM)+ Rotenone (0.02mM)+OG(0.2mM) (15mM) + Malate(15mM) (lSmM)+ Malate (15mM) -I-OG(0.2 mM) I t 30 60 MINUTES

Figure 4. Standard medium and conditions employed. Protein

Final

least

was .17 mg albumin/ml

localize

the site

DBI (3.0 mM) does not completely

conditione.

completely

blocka

The difference

and of ICI-Induced

and 0.58 mg mito-

system which cau8ea contraction. of interaction

part of the energy required

though it

was 3.0 mls.

protein-ml.

the energy transfer conclusively

in the text were

volume in spectrophotometer

concentration

chcldrial

as described

contraction

These data do not

of ICI but chow that

for contraction

comes from site

block the ICI-induced

ICI-induced

contraction

in susceptibility to inhibition

1057

uncoupling

at 2. even

under identical

of ICI-induced

uncoupling

by DBI presumably reflecte

Vol. 28, No. 6, 1967

BIOCHEMICAL AND BIOPHYSICAL

Effect

of

DBI

on

ICI-Induced

RESEARCH COMMUNICATIONS

Structural

Changes

I

Swelling

-0.0 ---0.2 ---3.0

mM mM mM

.

DBI DBI DBI

.

0.750-

,

6 t

,

.

I

* ‘I

Standard

Medium

Succinate

(45 mW

+

Rotenone

(0.02

‘.

‘\

I*..

mM)

pH 1.4 , 26.C 0.600,

1 30

0

I 60

MINUTES

Figure

5. Standard medium and conditions employed.

Final

concentration

as described

volume in apectrophotometer

in the text were MS 3.0 ml.

was 0.17 mg/ml albumin and .55 w/ml

FYoteln

mitochondrial

protein.

the action electron

of DBI ae an inhibitor transport

B&*-dependent, phosphorylation,

ae opposed to

per se.

The a111611amplitude concentrations

of energy transfer

contraction

of mitochondrie

of ICI is nub&rate-dependent occurs simultaneously la inhibited

1058

low

and to a less extent

with uncoupling

by octylguanidine

seen with

of oxidative

or DBI and does not

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 28, No. 6, 1967

Effect

of

DBI

on

ICI-

Induced

Structural

Changes

I -0.0

mM

DBI

---0.2

mM

DBI

- - - 3.0 mM

DBI

‘-.*

0

30

Swelling

*...

-..

60

MINUTES

Figure 6. Standard medium and conditiona employed.

Final

concentration

as described

volume in spectrophotometer

in the text were was 3.0 ml.

Protein

was 0.17 mg/ml albumin and .55 mg/ml mitochondrial

protein.

depend on eubatrate rotenone). tic

level

Thle contraction

of a high energy state

but actual

(occurs with

resembles that of mitochondria

volume change or .eXtrusion

not been demonstrated (Lehninger,

phosphorylation

comcanitant

1962; Ghappeland

described (Aezone

1059

as

and

characterie-

and Azzi,

1965),

of water fran mitochondria

with small amplitude

Greville,

auccinate

has

contraction

1963; Azzone and Azzi,

1965).

Vol. 28, No. 6, 1967

Therefore

BIOCHEMICAL

such contraction

contraction

seen after

Mitochondrial associated

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

need not be analogous to the large amplitude

ATP addition

swelling

to swollen mitochondria.

occurring

with inhibition

at high ICI concentration

of respiration

is

by ICI and is blocked but

not reversed by 4 mM ATP even in the presence of ollgomycln. swelling

is not influenced

trations

of ICI when both Mg* and substrate

by 4 mM ITP or GTP, occurs at lower concen-

medium, occur8 in the absence of substrate by oligomycin,

octylguanidlne,

are dissimilar

from those of large-amplitude,

(Chappell

and Greville,

concentrations

1963).

swelling

are omitted

Current

is facilitated

from the

and Pi, and 18 not blocked

DBI or Nag.

These characteristics Pi-induced

swelling

evidence suggests that

of ICI induce irreversible

and that this

The

swelling

high

of mitochondria

by a low energy state of the

mitochondria. Studies

are in progress to characterize

opposing actions

of different

changes in mitochondrla.

further

concentrations

The titration

the apparently

of ICI on structural

approach illustrated

in this

study should provide a good basis for comparison of the effects increasing

concentrations

of ICI and other compounds on swelling,

contraction

and oxidative

explanation

for dlscrepanciea

on structural

states

of

phosphorylation

and may provide an

noted in previous

of mitochondria

(Lehninger,

etudiee

of uncouplers

1962).

Azzone, G.F., and Azei, A., Volume Change8 in Liver Mitochondrla Proc. Nat. Acad. Sci., Chappel, J.B.,

U.S., 52, 1084 (1965).

and Greville,

G.D., The Influence

of the Suspending Medium on the Properties SOC. Symposia, 3,

39 (1963).

1060

of the Composition

of Mitochondria.

Biochem.

Vol. 28, No. 6, 1967

Prauklin,

BIOCHEMICAL

T.J.,

Jones, C.W., and Redfearu,

Thiophens on the Electron

Transport

of Respiratory

Activities

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

E.R., Rffect

of Substituted

and Adenosine Triphoaphataae Biochem. Biophys. Acta l.Jl,

Particlea.

240 (1967 a).

Franklin,

T.J.,

and Hlgginsou,

Cytotoxic

Imuuosuppreesive

B., Biochemical

Studies with a IVex

Agent, 3-Acetyl-5-(4-fluorobenzylidine)-

2,5-dihydro-4-hydroxy-2-oxothiophen

(ICI 47776).

Biochem. J., 102,

705 (196-r b).

Lehninger, Relation Peter,

A.L., to

Phoaphorylation.

Oxidatlve

J.B.,

Mitochondria

Water Uptake aud Rxtrusion

and Lee, L.D.,

Physiol.

Characteristics

(1967).

Preeman,

B.C., Specific Energy-linked

Inhibitors

Functions

of Skeletal

of Rnergy Tramfer,

of Mitochondria,

in

Rev., 42, 467 (1962).

Prepared by a IVew, Improved Technique.

submitted

editor,

by Mltochondria

Muscle

Science,

in B. Chance,

p. 181, Acad. Press,

New York (1963). Tata, J.R.,

et al.,

The Action

of Thyroid Hormones at the Cell Level.

Biochem. J., 86, 4c-3 (1963) Acknowledgements:

The ICI was kindly

provided by Dr. W. Hepworth.

Mr. J. Armstrong and Mrs. J. Payne provided aseietance.

Studies supported

excellent

technical

in part by RIR Grant RD 02584-01,

American Cancer Society Grant P 430.

1061