Influence of thyroid and growth hormone status on the rate of regulated Ca2+ efflux from rat liver mitochondria

BIOCHEMICAL

Vol. 108, No. 1, 1982 September

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages

16, 1982

307-314

INFLUENCE OF THYROID AND GROWTH HORMONE STATUS ON THE RATE OF REGULATED Ca2+ EFFLUX FROM RAT LIVER MITOCHONDRIA Roger

L. Greif, Gary Fiskurn?, Denise and Albert L. Lehninger*

A. Sloane

Department of Physiology University Medical College New York City 10021

Cornell

*Department of Physiological Chemistry Johns Hopkins University School of Medicine Baltimore, Maryland 21205 Received

July

28,

1982

SUMMARY: Measurements were made of Ca2+ efflux from liver mitochondria of normal, surgically thyroparathyroidectomized and hypophysectomized rats. Release of Ca2+ from preloaded mitochondria was initiated by the addition which stimuof ruthenium red, which blocks Ca2+ influx, and oxaloacetate, lates Ca2+ efflux by bringing the mitochondrial pyridine nucleotides to a The time required to release a small relatively oxidized steady-state. amount of Ca2+ from mitochondria isolated from thyroparathyroidectomized or hypophysectomized rats was far longer than that observed with mitochondria from normal animals. Treatment of such rats with either triiodothyronine or bovine growth hormone for 3-4 days resulted in restoration of Ca2+ efflux to a near normal level. INTRODUCTION Intracellular cellular

processes

stimuli is the

be affected

and often

an important acts

The distribution

(1).

liver, Both

Ca2+ plays

regulated uptake

in part and release

by circulating

'To whom correspondence Biochemistry, George D.C. 20037.

of

role

as a second intracellular

Ca2+ in mitochondrial

of Ca2+ by isolated of

a number

should be addressed. Washington University

regulation

messenger

by energy-linked

levels

in the

rat

liver

of hormones,

for

of many

extracellular

some tissues,

such

Ca2+ transport mitochondria including

Present address: School of Medicine,

as (2).

can epine-

Department Washington,

of

dehydrogenase; bGH, bovine ABBREVIATIONS USED: a-GPDH, alpha-glycerophosphate growth hormone. con, control; EGTA, ethylene glycol bis(B-amino ethyl 1 ,N i -tetraacetic ester)N,N,N acid; GH, growth hormone; HEPES, 4-(2-hydroethyl)-1-piperazinethanesulfonic acid; hypox, hypophysectomized; OAA, oxaloacetate; RR, ruthenium red; T3, triiodothyronine; TMPD, N,N,N1,N1tetramethylphenylenediamine; TX, thyroparathyroidectomized

0006-291X/82/170307-08$01.00/0 307

Copyright 0 I982 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 108, No. 1, 1982 phrine

and glucagon

transport state,

is

(3,4) influenced

as indicated

by the

(6)

and the

pyridine

nucleotides

hormones

profoundly

oxidized

state

sensitivity

(7,8).

modes of report

(T3)

(5).

Evidence rat

liver

state is

regulating

of some of these

presented

mitochondrial

NAD(P)H.

This

Ca2+

including

energy

Ca2+ efflux

of

the

here

provides

to adenine

intramitochondrial that

Ca2+ efflux

Ca2+ transport findings

Mitochondrial

factors,

of mitochondrial

oxidation-reduction

affect

RESEARCH COMMUNICATIONS

by many intracellular

of mitochondrial

and intracellular

AND BIOPHYSICAL

and triiodothyronine

also

nucleotides

preliminary

BIOCHEMICAL

thyroid

and growth

evoked

an example

by the

of how extra,

can be integrated.

has been presented

A

(9).

MATERIAL AND METHODS Animals and isolation of liver mitochondria. Male thyroparathyroidectomized rats (TX) and normal control rats (Con) were obtained from Charles River (CD strain) and weighed 50-70 gm on arrival. After bein maintained on a low iodine diet for 2 weeks, each rat was given 100 uCi 1 9 II intraperitoneally (i.p.> and thereafter kept on a normal laboratory chow diet for at least 6 weeks. Both TX and Con animals received 0.85% CaC12 in their drinking water. The TX rats weighed 140-195 gm when used and the Con rats 250-300 gm. Hypophysectomized (hypox) male rats obtained from the same supplier weighed 75-100 gm. They received no CaC12 in their drinking water and were used within 2 months of surgery. Target organs for pituitary hormones were markedly reduced in weight in these animals. T3 or bovine growth hormone (bGH) were dissolved in 0.01 N NaOH and administered i.p. Paired experimental and solvent injected control animals were fasted overnight before removal of livers and isolation of mitochondria. Mitochondria from the livers of the paired animals were prepared at the same time by the method of Schneider (10) in unbuffered 0.25 M sucrose. Mitochondrial protein was determined according to Lowry et al. (11) using bovine serum albumin as the standard.

Assays. Mitochondrial oxygen consumption was measured in a closed glass reaction vessel at 250C with a Clark oxygen electrode. Mitochondria were suspended at 1 mg protein/ml in medium containing 125 mM sucrose, 60 mM KCl, 3 mM K-HEPES (pH 7.1), 1 mM K-phosphate, 0.5 mM K-EGTA, 5 mM Ksuccinate and 4 uM rotenone. Respiratory control ratios were determined as described by Estabrook (12). Mitochondrial a-glycerophosphate dehydrogenase (a-GPDH) activity was determined by the method of Lee and Lardy (13) and served as a marker for the effect of T3. Total mitochondrial Ca present in 0.1 N HCl extracts of isolated mitochondria was determined by atomic of mitochondrial suspensions were absorption. Cpnges in th5+Ca2+ activity selective electrode (Radiometer F2112 Calcium measured at 25 C with a Ca Selectrode) using a Thomas combination glass electrode (Model 4094-L25) as reference. The output from the electrode was amplified through a SargentWelch High Impedance Accessory and fed into a Bausch and Lomb Model VOM 7 strip-chart recorder.

Materials. T3 was obtained Dr. M. Sonenberg, Sloan-Kettering fied according to the method of

from Calbiochem and bGH was the gift of Institute. Ruthenium red (RR) was puriLuft (14). 308

vol.

108, No. 1, 1982

BIOCHEMICAL

AND BIOPHYSICAL

I

I

10

5

I

0 t (mini

1

I

5

10

RESEARCH COMMUNICATIONS

I 15

I 20

Fig. 1 Respiration-dependent mitochond;$l Ca2+ uptake and oxaloacetatestimulated ruthenium red-insensitive Ca efflux. Rat liver mitochondria (mite) from either euthyroid control rats (con) or TX rats were added at a concentration of 2 mg protein/ml to 3 ml of medium containing 125 mM sucrose, 60 mM KCL, 3 ti K-HEPES, (pH 7.1), 0.2 mM K-phosphate, 4.0 mM _aycorbic acid, 4 uM rotenone, and 0.2 pM antimycin A. 20 nmoles of Ca2+-mg mitochondrial protein had previously been added in small aliquots to calibrate the response of the electrode. The suspension was maintained at 25OC, stirred magnetically, and continuously exposed to a gentle stream of humidified 02 to prevent anaerobiosis. Ca2+ uptake was initiated by the addition of the electron transport mediator TMPD (70 PM); efflux was initiated by the addition of 5 pM ruthenium red (RR) and 0.5 mM oxaloacetate (OAA). Measurements were made of the time taken after these additions for 10% of the added Ca2+ (2 nmoles*mg-l protein) to be released. The logarithmic response of the Ca2+ electrode was linearized graphically.

RESULTS The linearized cal

experiment

Ca2+ electrode in which

RR-insensitive

of a small

to a medium piratory

amount

containing

inhibitors

ficial

"site

tron

donor

most

of the

protein).

rotenone

ascorbate

Under

of Ca2+ uptake mone (GH) status

these

Ca'+*mg -'

rapid

conditions

TMPD in

uptake

observed

of the

of the

rats

(results 309

measured.

and

Transient

of mitochondria "site

1 and 2" res-

addition

of

the

arti-

presence

of

the

elec-

added

Ca2+ as well

medium

( % 2 nmoles*mg-l

in

initial

of different not

the

a typi-

Ca2+ influx

and the

Subsequent

no differences from

1 describe

upon addition

mediator

in

were

protein

as a contaminant

by mitochondria were

efflux

and antimycin.

resulted

Ca2+ present

Ca 2'

transport

Fig.

mitochondrial

of Ca2+ was observed 20 nmoles

3" electron

shown in

respiration-dependent

oxaloacetate-stimulated uptake

tracings

shown).

the

thyroid

rate

as

or extent

or growth

hor-

Vol. 108, No. 1, 1982

BIOCHEMICAL

Upon completion

of mitochondrial

itiated

by the which

(ON, chondrial After

addition

a lag

Ca2+ from

period liver

it

the

nearly

took

words,

for

oxygen

energy tive

was 3.7

and for

hypox

than

for

phosphorylation

animals

3.0

animals

and did

it

each not

described

that

average

the

mitochondria rat

liver

of

for

time

of TX rats

low mitochondrial

(n=17).

T3 and bGH treated differences

Ca2+ release

required

for

was more The

given

TX

CX-CPDH activity,

time in

release than

rats

In addition,

10% of

15-times

longer

had abnormally

310

and

from -t 0.8

oxidaCon

(n=17),

the mean RCRs for

of animals

from

the

with

(RCR) for

in Ca 2+ release obtained

given

permeability

was 4.4

Row A of Table

as expected

tested

mitochondria

of

also

were

ratio

groups

informais

membrane

it

T3 or bGH

animals, rat

control

TX animals

f 0.7

1 are

pertinent

each

mean RCRs for

for

with

the

by Fig.

mitochondria.

the

correlate

The mean values as those

The pooled

was 4.3

other

of paired

respiratory

mitofrom

with

group

generalized

was

between

injected

of each

in

to be re-

mitochondria

with

from

a paired

TX rat.

apparent

were

isolated

n=lO),

the

also

same

mitochondria

along

by the

(s.d.,

Ca2+ load

data,

differences

(12). f 0.7

from

these

mitochondria

as reflected

coupling,

mitochondria

of

approximately

from

liver

of

Ca2+

isolated

of

that

added

when the

and between

animals

efflux

However,

rat

were

(7).

net

within

the

mitochondria

characteristics

electrode

normal

animals

and similar

In addition,

1.

from

10% of

in Ca2+ efflux

A summary the

from

Con and hypox

rats

days.

concerning

in Table the

from

TX or hypox

tion

that

differences

isolated

several

than

released 1).

of mito-

reaction rapid

of mitochondria for

oxidization

10% of the

Ca 2+ being

Ca2+ efflux

faster

the

occurred;

longer

of Ca2+ was in-

and oxaloacetate

relatively

RR and OAA (Fig.

lo-times

efflux

dehydrogenase

min,

of Con rats

of

through

malate

0.5

contaminating

almost

Significant

either

the

the

Ca2+ influx,

of Ca"

made to a suspension

lo-times

chondria

blocks

approximately

addition

In other

leased.

via

+ the

were

TX rat,

nucleotides

mitochondria

after

additions

release

of

(=2 nmolesamg-1) 2.5 min

the

RESEARCH COMMUNICATIONS

Ca2+ uptake,

of RR, which

stimulates

pyridine

AND BIOPHYSICAL

was greater times.

experiments

such

1. These

indicate

Ca2+ load

from

than

that

for

low body

of hypothyroid

liver

normal weights

animals

and

(Table

w

11.5 1.2

+ f

f 2

+ f

k *

f +

2 k

5.0b 0.1

3.3b 0.2

8.0a 0.4

5.7b 0.1

5.7b 3.9

9.ga 3.9

t 11.2a f 0.4

for

6 5

5 3

6 7

rats

(AND

of

thyroid

0.45 6.47

0.12 5.27

0.70 4.58

0.13 1.16

0.13 0.13

0.76 4.94

0.26 1.19

f 0.13 + 0.74

t 0.01 t 0.64

f 0.20 f 0.41

+ 0.02 + 0.16

+ 0.04 +I 0.04

rt 0.16 f 0.44

f 0.08 + 0.26

aGPDH Activit

different

and growth

15.1 18.1

21.0 30.0

18.8 18.7

22.7 22.6

11.6 14.8

20.8 20.2

12.6 23.3

hormone

A 0.5 2 0.4

f 3.1 f 5.1

k 2.3 + 3.2

+_ 4.1 f 1.6

f 0.7 f 1.3

-t 2.2 ? 2.1

+ 0.9 + 1.2

status.

animal pairs used for each study. Treatment of the rats and Methods section. All values listed are the means + the taken for release of 10% of the added Ca2+. on signed ranks distribution (19).

90 f 11 89 ?r 10

792 78+

842 35+

70% 1 224 2 13

138 f 15 138 f 9

165 + 141 +

125 f 5 278 f 29

Body Weight (gm!

data

in parentheses are the numbers of in the text and in the Materials The Ca*+ release time is the time of differences in data was based

Hypox + bGH (5) Hypox + bGH + T3

G

The numbers was as described standard error. Significance ap < .02 bp < .04

(5JHypOX Hypox + T3 (low)

F

11.4 2.1

29.2 1.6

(6) Hypox

E

Hypox + T3

20.2 1.3

23.6 10.2

16.7 5.4

42.4 1.8

Time

and accompanying

Ca*+ Release (min)

times

HYPOX (5) Con

+ bGH

+ T3

Animals

Ca2+ release

D

@):

(@:

B

C

(6$En

A

Paired

Mitochondrial

TABLE 1

Vol. 108, No. 1, 1982 1).

The endogenous

that

of mitochondria

from

other

data

ed in most and is

of

thus

BIOCHEMICAL mitochondrial from

the

paired

where

injection

of

for

of

resulted

(TX rats

ference but

3 days receiving

Since

of

chondria

TX rats

from

(stimulation

of

considerably animals,

Ca2+ efflux,

which

could

(not

the

significance

shown), As would

from

hypox

have

intact

rat

liver

drial

be expected which

rats,

(Table

of hypox

was very

low,

of

1.0 ug T3-gm-'

of

endogenous

dria.

for

Injection

of

the rat

(Row E). Ca but

very

similar

a much smaller

did

This

the

to that amount 312

by mitochonvariation

in

levels

but

endogenous

which

of

the

did

the hypox

a-GPDH animals.

by administration not

in a substantial with

to normal mitochon-

controls,

increased

observed

isolated

compared

for

treatment

result

Ca2+ load

hormones

slowly

state

was markedly

re-

observed

mitochondria

same as that hypothyroid

the

uncertain.

as other

Although

was the

of

the

mito-

of TX

iodothyronine

results,

1, Row D).

the hypox

mitochondrial to a level

is

Ca2+ very

rats

3 days

time

finding

from

to that

exhibited

by plasma

the

Treatment

of day-to-day

released

reflecting

of

because

previous

of GH from

10% of

that

GH and T3 as well

glands,

mitochondria

The a-GPDH activity

release

lack

the

output

at which

but

of this

Ca.

was similar

than

dif-

a-GPDH activity

in a shortening

less

release

no significant

1, Row C).

which rate

the

made of Ca2+ efflux

resulted

be explained

from

parathyroid

Ca content

activity

not

were

apparent

ngmgm-l-day-l)

increased

the

bGH (Table

the

were

of

mitochondrial

to increase

4 days)

cases

untreated

also

endogenous

Ca2+ efflux)

In both

was still normal

treatment

given for

shortening showed

evident

Row B of Table

of T3(1.0

3 days

50%

Ca was unchang-

differences.

doses

measurements

were

daily

was released

of

(15),

that

these

than

as is

in Ca 2+ efflux

high

less

T3 is known

TX rat

T3 treatment.

dria

level

bGH (1 i.u.

time

with

the

of

with

lease

on the

However,

in a significant

The 3-day

administered

pituitary

for

was approximately

mitochondrial

differences

with

T3 for

in Ca2+ efflux.)

had no effect

rats

TX rats

animals.

endogenous

to be responsible

RESEARCH COMMUNICATIONS

of TX rats

euthyroid 1, the

experiments

1 shows that

time.

BIOPHYSICAL

Ca content

shown in Table

unlikely

a period

AND

affect

the

decrease normal

of T3 (50 ng*gm-l-day-l

level in the

mitochonfor

4 days)

Vol. 108, No. 1, 1982 also

resulted

BIOCHEMICAL

in substantial

AND

BIOPHYSICAL

stimulation

RESEARCH COMMUNICATIONS

of mitochondrial

Ca2+ efflux,

as a-GPDH (Row F).

Administration

of T3 also

produced

tion

in bGH-treated

hypox

(Row G).

of Ca2+ efflux

rats

as well

an additional

accelera-

DISCUSSION The results hormone

of

status

of

mitochondria.

this

the

rat

Despite

stimulated,

studied

individual

experiments

endogenous

or administered

mitochondrial

Ca2+ in

glucagon ed at of

(3). least

perhaps

is

oxidation

not

known.

of

the

time-course

shown)

with

ani-

of

the

indicated

regulated

in

that

release

direction

from

of

the

Ca2+ retention

of

the

rapidly-

observed

agonists

by T3 described

T3 and GH accelerate oxidized

and

here

requir-

stimulatory

effect

release

of mitochon-

oxidation

described

(NAD(H) by Figure

oxidation

unpublished

observations). CMA-initiated

for

(17).

of

+ NADP(H))

Recent

have

mitochondria Further

evidence

than

from tests

will

of mitochondrial 313

indicates

total

course

a similar

normal

of

that to

However,

mitochondrial

of experiments rate

and TX rats

be necessary

mito-

due primarily

NADH (18). of

the

indicated

T3 ( and

of OAA is

redox-state during

that

pyridine

due to inhibition

by addition

the

mitochondrial is

of NADPH (16),

NADPH rather

1 (8)

oxidation

of the

possibility

Ca2+ efflux

determinations

nucleotides

the

state

One interesting

the

OAA-induced

whether

Ca2+ efflux

intramitochondrial

spectrophotometric

as that

obtained

of a-adrenergic

of

by a relatively

of mitochondria

pyridine

differ

mitochondrial

NADH-NADP+ transhydrogenase

stimulation the

exposure;

of OAA-

rats.

administration

by which

GH) potentiate

chondrial

for

rate

to be determined.

Ca2+ evoked

nucleotides

capacity

liver

The results

(not the

of

here

in the

results

study

and growth

by isolated

consistent.

the

38 pairs

thyroid

variation

quite

during

the

of Ca2+ efflux

Ca2+ efflux,

described

in vivo -~

The mechanism(s) drial

of

The acceleration

GH has yet

both

T3 or GH accelerated 36 out

3 days of

rate

same day were

the

after

that

day-to-day

performed

in

minutes

the

insensitive

effects

increase

within

affect

red

on the

The hormonal

demonstrate

substantial

ruthenium

mal pairs

induced

study

such

and extent (G.

of

Fiskum,

to establish

NADPH per -se is

altered

by

Vol. 108, No. 1, 1982 the

T3 or GH status

chondrial

BIOCHEMICAL of

activities

of mitochondrial T3 and GH-induced logically-important

the

rat

which Ca2+. changes

BIOPHYSICAL

and whether

could

Studies

AND

in turn are

also

in mitochondrial

responses

to these

these

RESEARCH COAAMUNlCATlONS

hormones

affect

influence

the

currently

in progress

Ca2+ efflux

retention

represent

other

mito-

and release to determine

if

physio-

hormones.

ACKNOWLEDGEMENTS We are grateful to Dr. James Godbold for assistance in the statistical analysis of the data. Gary Fiskum was a Public Health Service National Research Service Awardee (1 F32GM07172-01) during the course of this study. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Rasmussen, H. and Goodman, D.B.P. (1977) Physiol. Rev. 57, 421-509. Carafoli, E. and Crompton, M. (1978) In: Current Topics in Membranes and Transport, Vol. 10 (Bronner, F. and Kleinzeller, A., eds.), Academic Press, New York, pp 151-216. Taylor, W.M., Prpic, V., Exton, J.H. and Bygrave, F.L. (1980) Biochem. J. 188, 443-450. Prpic, V. and Bygrave, F.L. (1980) J. Biol. Chem. 255, 6193-6199. Shears, S.B. and Bronk, J.R. (1981) FEBS Letts. 126, 9-12. Zoccarato, F., Rugolo,M., Siliprandi, D. and Siliprandi, N. (1981) Eur. J. Biochem. 114, 195-199. Lehninger, A.L., Vercesi, A. and Bababunmi, E.A. (1978) Proc. Natl. Acad. Sci. USA 75, 1690-1694. Fiskum, G. and Lehninger, A.L. (1979) J. Biol. Chem. 254, 6236-6239. Greif, R.L., Fiskum, G., Sloane, D.A. and Lehninger, A.L. (1980) American Thyroid Assoc. Inc., 56th Meeting, T-23. Schneider, W.C. (1948) J. Biol. Chem. 176, 259-266. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Estabrook, R.W. (1967) In: Methods in Enzymology, Vol. 10 (Estabrook, R.W. and Pullman, M.E., eds.), Academic Press, New York, pp 41-47. Lee, Y.P. and Lardy, H.A. (1965) J. Biol. Chem. 240, 1427-1436. Luft, J.H. (1971) Anat. Rec. 171, 347-368. Hervas, F., Morreale de Escobar, G. and Escobar Del Rey, F. (1975) Endocrinol. 97, 91-101. Harris, E.J., Al-Shaikhaly, M. and Baum, H. (1979) Biochem. J. 182, 455-464. Hoch, F.L. (1976) J. Bioenerg. Biomemb. 8, 223-238. Vercesi, A. (1981) Fed. Proceed. 40, 1781. Lehman, E.L. (1975) In: Statistical Methods Based on Ranks, Holden Day, San Francisco, pp 123-132.

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