Changes in the sarcolemma of the hypothyroid heart

Vol. 80, No. 4, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 715-721

February 28, 1978

CHANGES IN THE SARCOLEMMAOF THE HYPOTHYROID HEART R.M.

Smith,

W.S. Osborne-White

CSIRO Division

of Human Nutrition,

Adelaide,

Received

December

and R.A.

'King

Kintore

South Australia,

Avenue,

5000.

5,1977

SUMMARY: Sarcolemmal membranes prepared from the hearts of thyroidectomised sheep showed a fall of more than 90% in both 5'-nucleotidase activity and the number of B-adrenergic receptor sites. A fall of more than 60% in both Na++K+ATPase and adenyl Either long-term or shortcyclase activities also occurred. term treatment with thyroid hormones brought about concerted recovery of these pairs of sarcolemmal functions. INTRODUCTION Several

of the physiological

hypothyroidism oxygen

such as the

consumption

and myocardial skeletal

of

tissues

(4,s)

may be interpreted membranes of

(3),

in terms

depresse'd

responsiveness (7).

This

could

g-adrenergic

an impairment

of the

cyclase

attributes

to an effect

on the

metabolic

lowered

rate

cardiac

impaired rate

and

output of

lipolysis

(6)

of

cells.

Two lines

relates

occur

of thought

some of the tissues

either

effects to

through

on the

cell

(6,

calorigenic

(2)

performance

have

to a

adrenergic a reduction

surface

in

or through

catecholamine-responsive

membrane and some evidence

the

in

changes in the plasma

effects

activity

occur

certain

of the

cell

each of these

of reasoning

the

receptors

activity

in the

the

of hypothyroid

the number of

adduced for

of

of these

basal

decreased

thyroid-sensitive

The first

adenyl

(11,

and the

emerged.

stimulation

depressed

contractility

muscle

changes that

has been

The second line

8, 9). effect

of thyroid

of the Na++K+ATPase in the

hormone cell

0006-291X/78/08Ok-0715$01,00/G 715

Copyright All rights

0 1978 by Academic Press, Inc. of reproduction in any form reserved.

BIOCHEMICAL

Vol. 80, ~NO. 4, 1978

membrane. maintain

Acting

as a sodiumion

intracellular

Na'

appropriate

to the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

pump this

enzyme helps

and K' concentrations

cellts

function.

Its

at levels

activity

in a number

of tissues

has been shown to depend upon the thyroid

the

(10-12)

animal

consumption

and to contribute

in the

is

points

of view.

and Williams recently

function

changed show features

et al.

sensitivity (8)

of

prepared

treatment

relatively

large

of hypothyroid activity

rats

the

thyroid

intracellular

thyroid

sarcolemmal

membranes prepared

In addition

measurements

as a marker

for

(7) have

receptor of

following

hormone.

On the treatment

increases

in heart

sheep to study functions

sites

rats

membrane preparations

the

but

from ventricular

heart

the also

muscle. effects

and activities

were made of the activity

sarcolemmal

these

(13)

hormone not only

on these

of

15) have shown that

K+ concentration

hormone status

thyroid

the heart

and Marinetti

doses of thyroid

We have used thyroidectomised of

of

energy

agonists

from the hearts

of Na++K+ATPase in heart

increases

both

hyperthyroidism

and Edelmari (14, with

with

number of $-adrenergic

fractions

hand Philipson

to

when the

to B-adrenergic

in particulate

other

occur

consistent

and Ciaraldi

shown an increased

with

that

Under conditions

shows an increased

substantially

status

cell.

The changes in heart status

to

in

muscle.

of 5'-nucleotidase

purity,

MATERIALS AND METHODS Fifteen Australian Merino wethers (body weight 24.2 z 0.4 Kg) were surgically thyroidectomised at 8 months of age and maintained in roofed individual pens until killed 5-6 months later, Eight weeks after successful thyroidectomy (as determined by plasma thyroxine) five animals were selected at random and treated with L-thyroxine (0.5 mg suspended in 0.5 ml peanut oil injected subcutaneously) every second day until they were killed. A further control group of 5 normal wethers of the same age was maintained under similar conditions. All animals were fed ad lib. a diet of wheaten hay chaff and dried lucerne chaff (3:l by weight). Three days prior to killing five of the untreated thyroidectomised

716

Vol. 80, No. 4, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

injection of 0.5 mg L-trianimals received a single intravenous and administered in phosphateiodothyronine freshly dissolved, At the time of killing plasma thyroxine levels buffered saline. in both untreated thyroidectomised animals and those that had been treated 72h earlier were CO.2 ug/lOO ml. In the animals treated continuously with L-thyroxine, plasma thyroxine (mean 5 SEM) was 11.2 l 1.4 ug/lOO ml, and in the normal animals it was 8.1 * 0.5 ug/lOO ml. Animals were killed by captive-bolt pistol, a sample of ventricular heart muscle quickly removed, minced and chilled and sarcolemmal membranes prepared by the method of Kidwai (16). Specific binding of (-)-3H-dihydroalprenolol to S-receptors was measured in duplicate samples of about 100 ug of sarcolemmal protein as described by Ciaraldi and Marinetti (13) except that the concentration of (-)-3H-dihydroalprenolol was 20nM. 5'-Nucleotidase activity was measured by the method of Aronson and Touster (17). Duplicate tubes containing about 25 ug sarcolemmal protein were incubated for 30 min. and 60 min. and release of Pi measured. Na++K+ATPase was measured at pH 7.4 in volumes of 1.0 ml containing NaCl (lOOmM), KC1 (20mM), MgC12 (3mM), EDTA (lmM), tris-ATP (2.5mM), tris-chloride buffer (50mM) and about 20 ug of sarcolemmal protein. Duplicate tubes were incubated at 370C for 30 min. and 60 min. with and without 10m4Mouabain. Reactions were stopped with 2.0 ml cold trichloroacetic acid (7.5% w/v) and Pi measured by the method of Fiske and Subbarow (18). Adenyl cyclase was estimated in duplicate at 30°C and pH 7.5 in final volumes of 0.2 ml containing trischloride buffer (50mM), MgC12 (5mM), theophylline (lOmM), NaF (5mM) ATP (2mM, in the presence of creatine kinase and excess creatine Reactions were phosphate) and about 60 ug of sarcolemmal protein. started by adding ATP (together with its regenerating system) in of the tubes a volume of 0.06 ml following 30 min. pre-incubation adding ATP the reaction was stopped at 300C. Ten minutes after The supernatant water bath. by heating tubes for 3 min. in a boiling to estimating cAMP.by means was chilled and stored at -200C prior of kits obtained from the Radiochemical Centre, Amersham, U.K. Protein was estimated by the method of Lowry et al. (19). Measurement of all sarcolemmal activities was carried out on the day Plasma thyroxine was measured by radioimmuno assay of killing. using kits from the Radiochemical Centre, Amersham, U.K. RESULTS AND DISCUSSION The results

are

set out

where group means (and SEM) for have been plotted activities expressed very

close

capacity activity.

as ordinates

graphically the three against

(and SEM) of 5'-nucleotidase; on a protein linear of the

basis.

correlation sarcolemmal

Reference

to the

in Figs,

Fig.

activities

results

mean

have been

1 shows an unexpected the specific

membranes and their

717

measured

the corresponding all

between

group

1 and 2

treatments

but

B-binding 5'-nucleotidase

indicated

in Fig.

1

BIOCHEMICAL

Vol. 80, No. 4, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I b/ I

350

-4

N0Ullal

-4

300

G E 200 2 E ” P z iLy Q 100

01

0

I

1

100

200

5’Nucleotidase

(nmol/mg

protein

per

02

mln)

I

0 5’Nucleotidase

100 (nmol/mg

protein

,

1

200 per

250

FIGURE 1. Specific binding of (-)-3H-dihydroalprenolol to indicate the number of B-adrenergic receptor sites on sarcolemma from sheep heart plotted against 5' -nucleotidase activity of the same preparations. Treatments of the various groups are indicated on the figure near the appropriate values which show mean fSEM for the two activities for the group concerned. TX = untreated thyroidectomised animals; TX + T3 (72 h) = thyroidectomised animals treated 72 h previously with 0.5 mg L-tri-iodothyronine; TX + T4 = thyroidectomised animals treated continuously for 3-4 months with the equivalent of 250 ug of L-thyroxine per day; NORMAL = normal controls. Replication was 5. FIGURE 2. Activities of Na++K+ATPase (open circles) and adenyl cyclase (closed circles) plotted against 5'-nucleotidase activities in sheep heart sarcolemma. Treatments and replication as in Figure 1.

shows that (by

more

both than

of these

90%) by thyroidectomy

were substantially physiological response hours

and equally

were very and that

restored

doses of L-thyroxine.

of both

earlier

activities

with

These results

activities a single

dose of

might

be interpreted

718

both

but

in animals

depleted

activities

by treatment

A smaller

was evident

severely

with again treated

tri-iodothyronine. to mean that

the

equal 72

min)

BIOCHEMICAL

Vol. 80, No. 4,1978

sarcolemmal larger

preparations

quantities

of true

did preparations that

from the

5'-nucleotidase

on the rat

specific

heart

effect

since

however, both

marker

to believe

shown in Fig.

together

5'-nucleotidase If

the origin.

attributable

to

lemma1 protein,

activities

of both

body of

evidence

tissues

including

(6,

hypothyroid that

that

is

7, 9). hearts

also

sarcolemmal

they

that

13).

same

There

is,

of

activities

heart.

adenyl for

The preparations

scales)

do not pass through that

with

2 that

the hypothyroid

cyclase is

as compared now a substantial in several

animals

(10-12,

activity,

activity

of sarcolemma

showed no indication

in the

falls

cyclase its

is

non-sarco-

increase

There

of the

719

14, at

15)

least

upon thyroid from our

gross

contamination

to have caused a ten-fold The material

concerted

the

B-binding

protein

five-fold

dependent

protein.

from

in activity

the proposition

of hypothyroid

would seem to be necessary

of true

(8,

(on appropriate

Na++K+ATPase activity

the heart

sites.

from the

cyclase

from Fig.

from normal

some evidence

in some tissues, hormone

fall

Na++K+ATPase and adenyl

sarcolemma

is

b-binding

and specific

follows

shows an approximately

and there

the

of sarcolemmal

it

of

fractions

but

activity

sarcolemma

the

activity

1 is real.

we are to accept

then

be argued

of particulate

when plotted

dilution

then

hormone treatment

that

activity,

in 5' -nucleotidase

than

thyroid

2 Na++K+ATPase and adenyl

are seen to fall

with

specific

of

capacity

normal

enzymes were not measured

good reason

In Fig.

fall

of effects

could

much dilution

protein

then be supposed to have suffered

parameters

against

It

contained

numbers of

B-binding

might

hearts.

sarcolemma

demonstrations

contained ten-fold

of non-sarcolemmal normal

and normal

animals

to cause a further

protein)

the hypothyroid

Previous

from hypothyroid

(sufficient

sarcolemmal

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is recovered

dilution as a

Vol. 80, No. 4, 1978

BIOCHEMICAL

discrete

and visible

gradient

and it

soluble

TX + T4,

band from the topmost

is subsequently

protein.

the various

Recoveries

treatments

1,4?0,1

These values

indicate

mg; TX + T3 (72 h),

therefore

sarcolemma

to treatment

of both

changing

may be significant

adenyl

cyclase

are directed

agonists

(20)

toward

still

very

1.9zO.3

activities

whereas both

in

The results

also

fall

cyclase.

in the The

activities

of considerable

interest.

Na++K+ATPase and

enzymes whose active

cytoplasm,

to reside

in

the active

sites

for

centres

site

of

8-adrenergic

on the outside

surface

the plasma membrane.

ACKNOWLEDGEMENTS We thank thyroidectomies thyroxine.

Dr. G.H. McIntosh for and Miss D.M. Martin

carrying out the surgical for assays of plasma

REFERENCES 1.

Barker, 170,

2.

Strauer, B.E. and Schulze, 71, 624-644.

S.B. and Klitgaard, 81-86.

mg.

specific

responded

of sarcolemmal is

both

sharply

substantial

and the binding

believed

for

1.52 0.1 mg;

that

fell

status

the

fraction

mg; Tx,

hormone,

pairs

thyroid that

inward

are both

normal, 1.7TO.2

both

thyroid

these

in this

contamination,

are plasma-membrane

5'-nucleotidase

protein

Na++K+ATPase and adenyl

way in which

of a sucrose

from any contaminating

activity

and that

with

portion

seems justified

a more modest although

changed with It

gross

and 5'-nucleotidase

activities cohesive

of total

do not suggest

hypothyroid concert

freed

were as follows:

The conclusion B-binding

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

H.M.

(1952)

W. (1976)

720

Am, J.

Basic

Physiol.

Res, Cardiol.

of

Vol. 80, No. 4, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3.

Buccino, R.A., Spann, J.F., and Braunwald, E. (1967)

4.

Lambert, (1951)

5.

Gold, H.K., Spann, J.F. Invest. 49, 849-854.

6.

Krishna, Acad.

7.

Guttler, R.B., Otis, C.L., Shaw, J.W., Warren, D.W. and Nicoloff, J.T. (1975) in Thyroid Hormone Metabolism, Ed. by Harland, W.A. and Orr, J.S. pp 201-211 Academic Press: London.

8.

Williams, L.T., Lefkowitz, D..R. and Besch, H.R. Jr. 2787-2789.

9.

Will-Shahab, Acta biol.

10.

Ismail-Beigi, F. and Edelman, hi, USA 67, 1071-1078.

I.S,

(1970)

Proc.

11.

Ismail-Beigi,

1,s.

(1971)

J.

57,

Pool, P.E., Sonnenblick, E.H. J. Clin. Invest. 46, 1669-1682.

E.H., Underdahl, L.O., Beckett, S. and Mederos, J. Clin. Endocrinol. 11, 1186-1205.

G., Sci.

Jr.

and Braunwald,

Hynie, S. and Brodie, 59, 884-889.

B.B.

E.

(1970)

(1968)

Proc.

R.J., Watanabe, (1977) J. Biol.

and Edelman,

J. Nat.

I.

(1976) Nat.

Acad.

Gen. Physiol.

710-722.

12.

Lo,

and Edelman,

13.

Ciaraldi, T. and Marinetti, Res. Commun. 74, 984-991.

14.

K.D. and Edelman, Philipson, 232, C196-C201,

I.S.

(1977)

Am, J.

Physiol.

15.

Philipson,

I.S.

(1977)

Am. J.

Physiol.

c-s. 7840.

K.D,

232,

C202-C206,

16.

Kidwai,

A.M.

17.

Aronson, 3lA,

18.

Fiske, 66,

Clin.

A.M., Hathaway, Chem. 252,

L., Wollenberger, A. and Kittner, med. germ. 35, 829-835.

F.

L.O.

I.S.

G.V.

and Edelman,

(1974)

Methods

N.N. and Touster, 90-102. C.H.

(1976)

and Subbarow,

J.

Biol.

(1977)

Chem. 251,

Biochem.

in Enzymology

0. Y.

(1974) (1925)

31A,

Methods J.

Biol.

7834-

Biophys.

134-142.

in Enzymology Chem.

375-400,

19.

Lowry, O.H., Rosebrough, N.J., Farr, A.L. (1951) J. Biol. Chem. 193, 265-275.

20.

Frick, G.P. and Lowenstein, :251. 6372-6378.

J.M.

721

(1976)

and Randall, J.

Biol.

Chem.

R.J.