Restorative effects of spermine on oxidative phosphorylation and respiration in heat-aged mitochondria

Vol. 108, No. 1, 1982 September

AND BIOPHYSICAL

BIOCHEMICAL

RESEARCH COMMUNICATIONS Pages

16, 1982

174-181

RESTORAT IVE EFFECTS OF SPERMINE CN OXIDAT IVE PHOSPHORYLAT ION AND RESP IRAT ION IN HEAT -AGED MITCCHONDR IA J.E.

Phillips

and R.R.J.

Department University Santa Barbara, Received

July

Chaffee

of Ergoncmics of Califcmia Califcmia 93106

27, 1982

Samples cf fresh rat liver mi tcchcndria were heat-aged in 37 ‘C sucrose sufficiently to cause approximately a 33% reduction in the ADP :0 ratio when using beta-hydroxybutyric acid as substrate. Subsequent pclarographic assays showed spermine concentraticns between 0.0157 and 0.250 nM tc cause a striking linear restoration cf the ADP:O ratic. Spermine also improves both the respiratory rate during the conversicn cf ADP to ATP and the respiratory ccntrcl ratio cf heat-aged mitcchondria. The effects cf spermine on the respiratcry rate after ADP tc ATP conversion vary depending cn the spermine ccncentrat ion. Spermine has no significant effects on mitcchcndrial respiration prier tc additicn cf ADP. SJWARY :

isct

INTRODUCT ION In

the

past

many

stabilize,

prct ect

and

deleterious

agents

(see

mitcchcndrial have

preserve l-3).

membranes

also

presented

cf

mitcchcndrial

in

that

and

investigators

they

their

by

that

hcmologues,

the

publications

in

cf polyamines

cn mitochcndrial

tc

studies

significance

in

this

concent

bacteria

play

mitcchcndria.

All

labcratcry

(7-10).

they

performed

were

rat icn

These

174

on

rat

are

the

investigate

nc

the range

cf

detailed effects

ive prccesses

liver

rate

in bacteria

tc

were

igat crs

interesting

rcle were

studies

cf

invest

there

designed

against

change

studies

and phcsphcrylat

of l&g++ within

0006-291X/82/170174-08$01.00/0 Copyright 0 I982 by Academic Press, Inc. All rights of reproduction in any form reserved.

these

can

stabilization

and/cr

However,

ive

cells

Scme

a physiclcgical

specifically oxidat

the

(4,s). induce,

pclyamines

other

described

polyamines (6).

that and

pclyamines

polyamines

literature

because

react icn mixture

that

pcstulated

have

certain

cscillations

indicate

the

varicus Others

evidence volume

have

previous particular

mitcchcndria,

cf the calculated

at free

a

Vol. 108, No. 1, 1982

Mz++levels the

in

masking

BIOCHEMICAL the

effects

cytoplasm which

of

occur

@f % ++ in the mitcchondrial In the in

research

isotonic

sucrose

reductions of

these

studies our

in

the

research

to

protective

in

we have

and

spermine whether

such heat-aged

(11,121.

higher

This

obviated

unphysiclcgical

heat-aged time

rat

ive

levels

about

used

cn these

capacities.

polyamines

might

on

for

significant Sarrples

pclarographic

We have have,

oxidat

mitcchondria

capaci t i es.

subsequently

effects

liver

to bring

phcsphcrylat

were

restcrative

cell

one uses

sufficient

ive

of

determine

effects,

capacities

for

liver

RESEARCH COMMUNICATIONS

mixture.

mitcchondria

effects

rat

when

study

oxidat

heat-aged on

this 37°C

their

the

reaction

for at

AND 8lOPHYSlCAL

ive

in

and

designed

addition

to

phcsphcrylat

ive

mitcchondria. METHODS

Mi t ochondria were isolated from livers of adult n-ale Sprague-Dawley rats by the methcd described by Hcch and Lipmann (13). All assays were made at 37”C, using the pclarcgraphic apparatus described by Estabrook (14). In the liver in v‘ivc, acetoacetate is reduced to BOH (151, but if there is an excess cf BOH in vitro, the react ion is reversed and the oxidation of NADH thus generated can be followed pclarographically. For assaying BOH oxidation, a mcdif icat icn of the react icn mixture of Harris et al. (16) was used. The 3.6 ml reacticn mixture contained: 900 PMoles sucrose, 90 VMoles KCl, 18 ~Moles KH,PO, , 72 PMcles Tris-HCl, 1.08 PMoles ADP, 90 $vloles BOH and the apprcpriate concentrations of spermine and Iv&++. In each concentration et al. (17).

assay, 2-4 mg of mit cchondrial was determined spectrcphctcmetrically

Reduct ion was accomplished mately 3% of of the reaction Spermine concent rat icn hepatic levels statistically tlsing analysis

prctein

was used. the method

by

cf

Prct ein Lowry

of

mi t cchondrial phcsphcrylat ion efficiency and respiration in vitro by aging the mitcchondria at 37°C until approxithe phcsphcrylat ive efficiency was lest. The temperature mixture was rraintained at 37“C. concentration was varied between 0.0157 and 0.50 nM and I$‘+ was maintained at 0.93 IT&I, which is within the estimted of unbound cytoplasmic I@++ (11,121. Data were analyzed by correlated T test and further verification was cbtained cf variance with the Newman-Keuls Post Hoc Test. RESULTS

Figure f rem

cur

are put there is

into is

not

in

a

1 shows

the

approximate

vitro

heat

aging

reaction linear

statistically

mixtures increase different

3m

loss

regimen.

the from

the

When

containing in

in

0.0157, ADP :0 control 175

rat ic

ADP:O ratic

which

results

these

heat-aged

mitochondria

0.0628

and 0.250

rt%l spermine

back

value,

to i.e.

the

point neither

where E nor

it F

Vol. 108, No. 1, 1982

2.2

& a ;

2.1

kl

2.0

BIOCHEMICAL

A-B.. ........ B-C ......... A B-D (C&D)-(E&F) (E&F)-A

AND BIOPHYSICAL

RESEARCH COMMUNKATIONS A-B .,..,_._._ B-D B-E.. D-E E-F............

Ps.01 pa.05 Pst.05 _, P 5.05 NSD

Ps.01 Ps.05 Ps .Ol PS.05 NSD

F

6-4

.$ L

1.9 85.5%

2? ;$ U-J-

1.8

:

1.7

-E 0 - 55% - 48% - 44.4%

Y F %

1.6

B 72%

E

1.5

I

NON-AGED

AGED

0.0157

01

0.0628

SPERMINE

0.50

0.250

Changes seen in in vitrc and

Fieure chcndria

vitro

are

Changes in

seen

statistically

different

from

can be a potent

polyamine

ency

mitochcndria.

in heat-aged As

the

shown

RCR*.

RCR.

of

due

result

results

also

in

to

tc

of

indicate

oxidative

aging

(mMolar)

mite-

that

spermine

phosphorylative

causes

partial

a

55% reducticn

restoration

a lo”/, recovery

tiereas

ef fici-

of

at

0.250

in

the

lost

and 0.50

a 22% to 25% recovery. that

improvements

increase

in

state

3 of

are

added.

All

a

statistically

state

3**

in

the

respiration.

aged

RCR are The

mitochondria

except

the

significant

due

restorative

as

lowest

largely

effects

increasing level

iqnwvement

stirmresult

concentrations

of in

to

spermine

state

(0.0157

3 rates

when

to the aged value.

Figure

“RCR: -

these

I

0.50

0.250

due

Ratic

the

vitro

about

of

ccqared

After

is

rate

r&J) produced

Thus

restoring

ians

the

spermine

Ccntrol

I

0.0628

efficiency aging aging

of spermine. A.

in

addit there

3 shows

in

an

IM

2,

is approximtely

Figure

in

in

Figure

0.0628

n&i there

lation

in

Spermine

At

Respiratory

additims

I

0.0157

SPERMINE

oxidative phosphcrylatim after additions of spermine.

in

and after

I

AGED

02

(mMolar)

Fieure 1: cf mitcchcndria 2:

NON-AGED

in

4 shows spermine vitro

aging

Respiratory

the

to have unusual rate

Control

**State

3:

Respiration

during

***State

4:

Respiratim

after

of

state

effects

on state

4 increases

Ratio conversion conversion 176

of ADP tc ATP of ADP to ATP

by

4*** 1%

respiration.

as ccrrpared

to

Vol. 108, No. 1, 1982

BIOCHEMICAL

A-B.. . B-C B-D ..,._____ B-E B-F ,_,_._._._

Ps.01 NSD Ps.05 Ps.05 PS.05

AND BIOPHYSICAL

100%

RESEARCH COMMUNICATIONS A-B.. B-C A-C B-E. A-E

15 c

........ ......... ......... ......... ............

Pr.01 PS.05 Ps.01 Ps.05 NSD 120%

65%

F .F ‘u e a

63% 60.6%

14

1 12% 1 10%

F 2

13

102% 100%

L

52% 94%

B 1 AGED

NON-AGED

03

I 0.0157

I 0.0628

SPERMINE

Figure version)

Changes to aging

non-aged

aged

(fresh

AGED

0

0.0157

4

seen in mitochondrial of mitochcndria in

mi t cchcndria.

by an additional

the

NON-AGED

The

8%, but

mitochondria

in

show

mitochondrial)

the

respiration and after respiratim and after

presence presence

ADP-ATP ccnverof spermine.

KM spermine KM or 0.50

state

0.50

ADP-ATP conof spermine.

(after additions

of 0.0250 of

0.250

(mMolar)

(during additions

of 0.0157

a restoration

0.0628

SPERMINE

vitro

4: Changes seen in mitochondrial due to aging of mitochcndria in vitro

cent rol it

11

, 0.50

(mMolar)

3: due

Figure sion)

1 0.250

4 back

to

increases ti

spermine

the

non-aged

value. DISCUSSION

One

might

at

first

glance

polyamines

in

the

37’C

reaction

menbrane a

cr

generalized

herein

tiich present

ccncept

for by

5 or the

initiated

method

spermine oxidat of

in of

cn

the the

8.5%

state

results

are

stabilizing

mitochcndria sucrose

prevents

177

the

heat-aging results

further

procf

the

mitcchondrial

of

ive

and

thus

cent inued

this than

that

having

funct icnal

treatment.

rather

However,

study

tiich

a stabilizing

we

suppcrt ef feet

functions. by

nc doubt

3 respiration

as

Icngevity,

a restcrative

and phcsphorylat

heat-aging

6 minutes lcwering

has

ive

which in

based

our

mitcchcndrial

ef feet

arguments

on mi t cchcndrial Our

was

that

mixture

enhancing

prctective

degradation

the

pcssibly

interpret

by

exposing causes about

them

to

heat

darrage

as

is

50% and

increased

at

37°C

indicated state

BIOCHEMICAL

Vol. 10s. No. 1, 1982 4 by

about

ADP:O

ratic.

dria

are

12%

mixture

net

is

itself

ample,

respiraticn

or

minutes (which

shows

no

ing

the

period,

functions

the

in

in

slcpe

in

the

downward.

Subsequently

respiration

if

mixture.

NC such

state

37 ‘C

4

rates

stable-state

situaticn

measurements

wherein

not

tc

be

in the ccrrpariscns

in

reaction

line

slcpes

of

with

time.

This

restcrat

al so.

We interpret

this

effect

which

(the

pericd

which

time

mixture

is in the

in

state

state

ion

mitcchondria

to

any

there

is

I. in

before ccntact 178

with there

a rratter

cent inu-

a 3 minute a curving

the placed

in

ADP,

state

Thus is

serious

of

the

a rapid

of

less

tc

curving rat its

restorative than

a minute

ccnmence) and

errcr

straight

to ADP :0

is

the

added

shcw any

respect

spermine;

in

3

temperature

cf spermine in

the

pclarcgraphic

source

a change

4

a relatively

mixture

measurements with

were

reacticn

of

neither

apply

that:

completed

ccme in

reacticn

4 respiration:

chamber

pericd

each increment

assays

as evidence

react icn

the

reacticn

in

of

there

1

state

functions.

spermine,

one

during

are

additicn

line

this

with

appears

and

the

intrcduces

Thus,

successive

3 and

initiated the

that

made.

to

of

at

tc decline

for

the

ever

upward

ex-

treatment

25%, i.e.

mit ochcndria

throughcut

exposure

a variable

the

absence

cf about

ccntinue

straight

exists

heat

being

are

see,

Fcr

3 respi r-

aging

functions tc

run

in

in

metirane

state

3 minutes

expect

the

the

whether

sucrose

a curving

prior

the

which

tc once

occurs:

respiraticn mixture,

fcr

be

were

respiraticn

37’C

slope

should

ccntrol

curving

measured

cne would

there

,

the

3 respiration

mixture,

react icn

appears

state

ccnditicns

and

i en cf mi t ochcndrial

mixture,

metabclic

37 OC react ion and

degradat

react icn

a change

if

even

reacticn

pclarcgraphically. linear

ing

degradation

cf

pctarcgraph,

in

suppcrt

rapid

mixture

the

the mi tochcnof

a stabilization

the

in

of the

c.f this

ADP is

5 or 6 minutes

heat -induced 37 ‘C

cf

capable

temperature

we assayed of

a drcp

but when

reaction

cause

37 “C mixture

declines

37’c

the

addition

RCR) and

mixture

the

fact,

which

to

the

ccntinuance

apparently

prior in

If

in

the

change

mixture.

In

cf

react icn

a

RESEARCH COMMUNICATIONS

is permanent,

that

causing

spermine,

to

aticn

the

shcw indications

in

saris

damge

intc

funct icn.

respect

twc

put

they

mi t ochcndrial

themselves,

lcwering

scme of this

subsequently s,

with

a ccnsequent

Prcbably

ATP synthesi

of

(with

AND BIOPHYSICAL

2. the

during extent

Vol. 108, No. 1, 1982 of

these

BIOCHEMICAL

restcrative

effects

AND BIOPHYSICAL

appears

RESEARCH COMMUNICATIONS

tc be a functicn

cf

the

spermin.e

ccncen-

tration. It

should

effects

are

mere

be kept not

significant

cf

the

mitochcndrial

of

as

little

is

much

cellular

physiclcgical

as 0.0157

is

as

spermine

which

latter

nMoles/ml)

The

restcrat

represent

exquisite

in

the

with

tc

affect

studies

a much

tc

a change

herein

respect

to

presented

the

swelling

ive

sensitivity

perfcrrrance

shcwn

mi t ochcndria

and

would

phosphorylative

sensitivity

the

would

have

cellular

induced

which

indicates

stressful

as

changes

Unfortunately

pclyaminerepcrted

by

mi t ochondrial similar

studies

substitutes order

fcr of

enzymes

lives

tc

be

would

ccunteracted

asscciated (22-26)

from to

levels of

it

and has

animals

added

Thus

number

be

levels

evidence

isolated

any

This

of

spermine

with

changes

efficiency.

Furthermore,

to

might

cytcplasmic

shown

(9).

responsible

(18-21).

darrage in

polyamine

experiments

mechanisms

effect

the

half

pclyamines

been

not

in cells

presented

acclimated

polyamines is

environmental

than

inconceivable

m3y

a

dc mitc-

of animals

factcrs

tc

that

undergcing

cause

restorative

in vivo. our

specific

since

endocrinclcgical

contrcls

due

place

differently

intracellular

cn mitochondria

the

i.e.

mi tcchcndria

changes

specific

ion

been

(27).

frcm

in

physiclogical effects

have

respond

isolated

of

shcrt

ive phcsphorylat

responses

levels

changes

levels,

that

take

in

mitcchondrial

causing

responses

environment

chcndria

can

that

oxidat

polyamine

shifts

remarkably

pcssiblity

improve

rapid

cell

responses

stress

one

the

stabilizing

respcnse.

and

that

in

Scme anirral

what

the

evidence

the

cellular

rapid

but

(15.7

of

synthesis

int reduce

in

r&l

than

There

their

by

exclusive,

ccncept s of

(4-6).

such fcr

that

autually

stabilizaticn

ethers

mind

respiratory

greater

induced

in

s on

we have spermine nagnitutde

of

do not spermine

answer acticn

are

oxida t ive

and

phcsphcrylat

ccnducted

using

putrisine,

show cause

that the

none effects 179

of

these that

any

questicns

tiich ive

cause

concerning the

functions.

spennidine, at

concentrations

spermine

dces.

cbserved However,

cr

a++

as

within Thus

the

Vol. 108, No. 1, 1982

BIOCHEMICAL

simplistic

spermine

or

@g++

idea

that

substitute

Whatever

and

spermine

oxidase

system,

anywhere

near

acts

thus

is since

AND BIOPHYSICAL as a generalized

prcduces

its

doing

is

cbvious

our

results

such exquisite

RESEARCH COMMUNICATIONS

mst

using

respcnses

organic

effects

is

when

other

bivalent

certainly studies

net

cation tenable.

involve

substrates

have

the not

BOH shown

tc spermine.

ACKNOWLEDGEMENTS We wish technical

its

to

thank

grateful

suppcrt

for

respect

and Debra

K. Msttson

for

their

excellent

assistance.

We are

in Aging,

James R. Allen

This A M 18895-01,

work

the

at tendance

Washington to the

tc

research was

D.C.,

at Dec.

herein

for

the 19,

Integrative

Conference 1980,

which

Bianedical on

the

enriched

Role

Research cf

for

Mi t ochcndria

our perspective

with

presented.

supper-ted

and NASA-Ames

Fund

by

CNR Contract

NOOOl4-75X-0506,

NIH

Grant

NCA 2aR-608-601.

REFERENCES

1. 2. 3.

2: 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Cohen, S.S. (1971) Intrcduction to the Polyamines, Prentice-Hall, Inc., Englewcod Cliffs, New Jersey. Russell, D.H. (1973) Polyamines in Nonral and Neoplastic Growth, Raven Press, New York. Bachrach, U. (1973) Functicn of Naturally Occurring Polyamines, Academic Press, Inc., New York. Tabcr, C.W. (1960) Bicchem. Biophys. Res. Ccmnln. 2, 117-120. Herbst, E.J. and Witherspcon, B.H. (1960) Fed. Proc. 19, 138. Hunnan-Seppala, A. (1971) J. Bioenergetics 2, 197-207. Chaffee, R.R.J., Salganiccff, L., Arine, R.M., Rochelle, R.H. and Schultz, E.L. (1977) Biochem. Biophys. Res. Ccnmm. 77, 1009-1016. Chaffee, R.R.J., Arine, R.M., Rcchelle, R.H. and Walker, C.D. (1978) In.:. Advances in Pclyamine Research (-bell, R.A., Morris, D.R., Bartos, D., Daves, G.D. and Bartos, F., eds) 2, 123-128. Chaffee, R.R.J., Arine, R.M., Rochelle, R.H., and Walker, C.D. (1978) Experient ia Suppl . 32, 95-99. Chaffee, R.R.J., Arine, R.M., and Rochelle, R.H. (1979) Biochem. Biophys. Res. Cannm. 86, 293-299. Velosc, D., Guynn, R.W., Oskarsson, M., and Veech, R.L. (1973) J. Biol. Cl-em. 248, 4811-4819. Vander Meer, R., Akerbcan, T.P.M., Groen, A.K., and Toger, J .M. (1978) J. Biochem. 84, 421-428. HO&I, F.L., and Lipmann, F. (1954) Proc. Natl. Acad. Sci. 40, 909-921. Btabrook, R.W. (1967) In: Methods in Enzymology (Estabrock, R.W. and Pullman, M.E. eds) X, 41-47, Academic Press, New York. (1973) Regulation in Metabolism, pp. Newshclme, E.A., and Start, C. 324-325, Jchn Wiley and Sons, New York. (1967) Biochemistry 6, Harris, E.J., Catlin, G., and Pressman, B.C. 1360-1369. 180

Vol. 108, No. 1, 1982

BIOCHEM!CAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

17. Lowry, O.H., Rcsebrough, N.J., Farr, A.L., and Randall, R.]. (1951) J. Bicl. Chem. 193, 265-275. 18. Russell, D.H., and Taylcr, R.L. (1971) Endocrinology 88, 1397-1403. 19. Russell, D.H., and Potyraj, J.J. (1972) Biochem. J. 128, 1109-1115. 20. Kay, J.E., Lindsey, V.J., and Cocke, A. (1972) FEBSLetters 21 123-126. 21. Russel1,D.H.. Snyder, S.H., and Medina, V.J. (1970) Endocrinology 88, 1414-1419. 22. Levine, J.H., Learning, A.B., and Raskin, P. (1978) In: Advances in Pclyamine Research (Caqbell, R.A., Morris, D.R., Bartos, D., Daves, G.D., and Bartcs, F., eds) 1, 51-58, Raven Press, NewYork. 23. Ja"ga;;J., Raina, A., and Siimes, M. (1968) Biochim. Biophys. Acta 166, . 24. Levine, J.H., Nicholson, W.E., Liddle, G.E., and Orth, D.N. (1973) Endocrinology 92, 1089-1095. 25. Kobayashi,Y., Kupelian, J., and Ivhudsley, D.V. (1971) Science 172, 379-380. 26. Nichclson,W.E., Levine, J.H., and Orth, D.N. (1976) Endocrinology 98, 123-128. 27. Tsvetnenko, E.Z., and Shugaley, V.S. (1978) Byull. Exsp. Biol. Med.8, 2830.

181