Effect of thyroxine and thiouracil on the Mg++-activated ATPase on the rat myocardium

Life Sciences Vol . 3, pp . 431-440, 1964. Pergamon Press, Inc. Printed in the United States .

EFFECT OF THYROXINE AND THIOURACIL ON THE Mg++ -ACTIVATED ATPase ON THE RAT MYOCARDIUM* K . M . Wang and M . Benmiloud Department of Pharmacology, St . Louis University School of Medicine, St . Louis, Missouri (Received 27 March 1964) Introduction It has been shown that in the myocardium of rat with cardiac hypertrophy, induced by reduced atmospheric pressure and elevated environmental temperature there is an increase in protein content in various heart fractions,

This increase varies with the different

fractions being more marked in myoglobin, actomyosin and mitochondrial fractions (1) .

The increased protein in these fractions

may bear some relationship to the increase of ATPase activity in cardiac hypertrophy reported by Rossi and Dianzani (2) .

Hypertro-

phy of the heart has also been produced in rats treated with thyroid hormone (3), but no data on the ATPase activity were reported . The available data do not elucidate whether hypertrophy of the heart produced by reduced atmospheric pressure and elevated environmental temperature and by thyroxine results in, is caused by or occurs simultaneously with similar chemical changes .

The present

paper is concerned with the effect of thyroxine and thiouracil ~ vitro and in vivo on the jp3ff-activated ATPase activity of the *This investigation was supported by grants from the Missouri Heart Association and the U . S, Public Health Service (Grant HE 04532) . 431

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EFFECT OF TIiYR.OXINE AND THIOURACIL

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rat heart .

Materials and Methods In vitro study .

Male albino rats weighing between 275 and

300 gm were sacrificed instantly by decapitation; the hearts were removed imtaediately, dissected free of fat and connective tissue, and weighed on a Rollar-Smith micro torsion balance .

The heart

was then chilled with cold isotonic sucrose solution, quickly blotted with filter paper and minced .

Nine ml of cold 0 .25M

sucrose solution were then added to each gm of heart tissue and the myocardium was then homogenized in all-glass homogenizer . The hoanogenate was centrifuged at 700 x g for 10 min .

The super-

natant was collected, the sediment resuspended, recentrifuged twice more, and the washed sediment (Sediment I, containing mostly the nuclei) was collected.

The washings were combined with the

original supernatant (Supernatant I), centrifuged at 5,000 x g for 10 min and the supernatant (Supernatant II) separated from the sediment (Sediment II, containing mostly the mitochondria) .

The

sediment was re-suspended and re-centrifuged twice more, and the washings were combined with supernatant II, which was further centrifuged at 95,000 x g for 1 hr to give sediment III (containing mostly the microsomes) and supernatant III (corresponding to soluble fraction) . Mg+f -activated ATPase activity was assayed on these centrifugal fractions essentially according to the method of Pullman et al . (4) .

The enzymatic activity is measured by determining

the Pi release in the presence of an ATP-regenerating system,

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EFFECT OF THYROXII~TE AND THIOiTRACIL

using phosphoenol-pyruvate (PEP) and pyruvate kinase (PK) .

433 The

assay mixture contained, in a final volume of 200 N.].: Tris*acetate, pH 7 .4, 100 ~unoles ; MgCl2, 0,6 Eunoles ; ATP, 1 umoles ; PEP, 1 Nmole, and PK, 20 4.tg and an appropriate amount of each heart centrifugal fraction .

The assay mixtures were incubated at 30° for 10 min .

The reaction was halted by the addition of 200 Wl of 1096 trichloroacetic acid (TOA) and the precipitate was removed by centrifugation .

Aliquotes of 200 Ei1 were used for the Pi determination

by the method of Lowry and Lopez (5) . Various concentrations of thyroxine, l, 5, 10, 50 and 100 x 109 M, were added to the different cellular fractions of the normal rat myocardium and the different fractions were assayed for the Mgt-activated ATPase activity . In vivo stu

,

Male albino rats, initially weighing between

218 and 245 gm, were used .

All rats were fed the regular Purina

laboratory chow and were allowed to drink ad libitum . of rats comprised 5 to 10 animals . control .

Each group

One group of rats served as

A second group of rats was given L-thyroxine in its

drinking water, in the amount of 1 LLg/ml water for a period of 10 days .

A third group of rats was

given thiouracil in its

drinking water, containing 0 .196 of thiouracil also for a 10 day period .

At the end of this period, the rats were sacrificed by

decapitation .

Heart, thyroid, and adrenals were removed immediate-

ly, dissected free of fat and connective tissue, and weighed .

Assay

of Mg++-activated ATPase activity in the various cellular fractions *Abbreviation :

Tris (hydroxymethyl) amino methane

434

EFFECT OF THYR.OXIIJE AND THIOiTRACIL

of the myocardium was carried out,

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T'he assay method and separation

of various cellular components were similar to that discribed in the in vitro study .

Results In vitro Effect of Thyroxine As seen in Fig, 1 all the incubated centrifugal fractions of the myocardium showed an increased ATPase activity at a concentration of 1 x 10 9 M thyroxine,

An almost linear in

crease in Mgt -activated ATPase activity was found in the 5,000 x g sediment and 95,000 x g supernatant with increasing concentrations of thyroxine, whereas the 700 x g sediment showed a steady and relatively mild decrease in the enzymatic activity with increasing concentrations of thyroxine,

The 95,000 x g sediment

showed a sudden increase of ATPase activity at 5 x 10 9 M thyro-

W h

<+40 L F S Z +10

r V r

0

â -10 è

so

TFIYROXIN! (M :10 - 91

~oo

FIG, 1 In Vitro Effect of Thyro~xine on the ATPase Activity of Various Centrifugal Fractions of Rat Myocardium

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EFFECT Ok' THYRO~CII~iE AND THIOURACIL

435

xine, then a sharp decline in enzymatic activity resulted with increasing concentrations of thyroxine ; at higher thyroxine concentrations, i .e . 50 - 100 x 10-9 M, a slight inhibition in ATPase occurred as seen in Fig, l . In Vivo Effect of Thyroxine and Thiouracil It can be seen in Table I that the body weight and heart weight of thyroxine fed rata were slightly heavier than the control .

The thyroid and adrenal weights were the same in

both the control group and the thyroxine treated group,

In the

thiouracil treated rats, on the other hand, they was practically no difference in the body weight gain as compared with controls, but a decrease in heart and adrenal weight and a doubling in thyroid weight . The difference between the effect of thyroxine and thiouracil on the Mg{{ -activated ATPase activity of the myocardial centrifugal fractions is summarized in Table 2 .

Thyroxine raised the

ATPase activity in the particulate fractions of 5,000 x g and 95,000 x g sediments, i .e, the fractions corresponding to the mitochondria and microso~nes but left unchanged the ATPase activity of the soluble fraction (95,000 x g supernatant) and the 700 x g sediment, which consists mostly of nuclei .

In the thio-

uracil-treated rat heart fractions, only the 95,000 x g sediment fraction showed an increase in activity . The protein content per unit of tissue weight was the same in the control group, the thyroxine treated and thiouracil treated animals as indicated in Table 3 .

This would show that the increase

of rat heart weight produced by feeding thyroxine is associated

438

EFFECT OF THYROXINE AND THIOiTRACIL

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EFFECT OF THYROXII~iE AND THIOURACIL

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Table 3 Protein Content of the Heart Group Ex4eriment I Control L-Thyroxine treated Thiouracil treated Experiment II Control L-Thyroxine treated Thiouracil treated

Heart Wt, (mg)

ma protein mg protein heart 100 mg heart Wt,

(10)

864 .8f13 .1

222,6±4 .3

25,7±0 .5

(10)

900 .0±27 .7

223 .7±7 .2

24 .8±0 .4

(10)

808 .6114 .7

202 .5±6 .2

25 .0±0 .6

(5)

912,1±22 .6

201,6-E9 .8

22 .1±1 .4

(5)

965 .9+42 .4

214,4+7 .8

22 .2+1 .3

(5)

831 .1±13 .4

182,0±13 .0

21 .9±1,4

with concomittant increase in total protein .

Discussion Hypertrophy of the heart induced by exposure to elevated environmental temperature and reduced atmospheric pressure may result in considerable increase (50916) in heart weight (1),

In

the present study there was only a 696 increase in the heart weight of rats fed with thyroxine .

This may have been larger with higher

doses of thyroxine and longer periods of treatment .

In the present

experiment as well as Sabel and Cohents experiment (1), the total heart protein content paralleled the increase in heart weight . Thiouracil although it doubled the thyroid weight decreased very slightly the heart weight and protein content . Thyroxine in vivo is known to cause an increase of Mg~activated ATPase activity in the mitochondrial fraction of the liver (6),

In the present experiment a similar increase was

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EFFECT OF THYROXII~TE AND THIOiTRACIL

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observed in the cellular fractions of the heart especially the 5,000 x g sediment which roughly corresponds to the mitochondrial fraction .

The soluble fraction and the nuclei rich fraction

were low in ATPase activities, and were also unaffected by thyroxine feeding .

Thus it seems that changes in the ATPase activity

of hypertrophic heart, whether induced by elevated temperature and reduced atmospheric pressure or thyroxine occur under the same circumstances as changes in the mitochondrial protein .

Thio-

uracil slightly stimulates ATPase activity of 95,000 x g sediment . The in vitro studies of the present work show that a lower thyroxine concentrations, Mgt-activated ATPase activity is stimulated in all centrifugal fractions .

It has also been demonstrated

by Lardy et al (7) that thyroxine stimulates the liver mitochondria ATPase in vitro .

Furthermore, it is interesting to note that

radioactive thyroxine binding studies in skeletal muscle cell fractions by Tata et al .

(8) showed the highest amount of radio-

activity in cell sap (105,000 x g supernatant) and mitochondria, both of which showed marked activation of ATPase activity .

This

is in agreement with the present observation that there is a linear increase of ATPase activity in 5,000 x g sediment (mitochondria fraction) and 95,000 x g supernatant with increasing concentrations of thyroxine in the incubation medium . The effect of different concentrations of thyroxine on ATPase activity of the various centrifugal fractions indicates that these heart fractions react differently toward thyroxine .

These varia

tions presumably axe the result of physical difference of the ATPases in the different fractions .

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EFFECT OF THYROXII~TE AND THIOiTRACIL

439

Summary The effect of thyroxine and thiouracil on the Mg++ -activated ATPase of the rat myocardium has been studied in vivo and in vitro . In

iv tro at a concentration of 1 x 10-9 M, thyroxine stimula-

tion of the Mgt -activated ATPase in all cellular components has been observed .

At the concentration of 5 x 10-9 M, only the 5,000

x g sediment and 95,000 x g supernatant showed an increase of this enzymatic activity,

At thyroxine concentrations of 5 x 10-9 to

100 x 10-9 , and 50 x 10-9 to 100 x 10-9 M, inhibition was demonstrated in the 700 x g and 95,000 x g sediments respectively . ~n vivo male albino rats were given thyroxine (1 ~,g/ml) and thiouracil (0,1%) ad libitum in their drinking water for ten days,

The thyroxine treatment increased the heart weight

whereas thiouracil treatment reduced the heart and adrenals size and raised the thyroid weight .

There was a slight but not stat-

istically significant elevation in the Mg+{-activated ATPase activity of the 5,000 x g and 95,000 x g sediment of the thyroxine treated rat heart and in the 95,000 x g sediment of thiouracil treated rat heart,

Reference 1.

H. Sabel, and F . Cohen, Proc . Soc . Exiler, Biol . & Med . 99~ 656, (1958) .

2.

G .F . Rossi and M.A. Dianzani Mor, Sperimentale, l~ 378,

3.

V. Korenchevsky, 3 .K. Paris and B . Benjamin, J . Gerontoloav 5, 120, (1950),

4,

M.D, Pullman, H.S . Penefsky, A, Datta and E . Racker, J . Biol . Chem, 235 , 3322, (1960),

(1958),

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5.

O .H, Lowry and J,A, Lopez, J . Biol . Chem . 162 , 421,

(1946),

6.

G,F . Maley, Am. J . Phvsiol . 188, 35,

7.

H,A, Lardy, H, Wellman and G . Feldott, Abstr . 121st Meeting Amer . Chew. Soc ., p . 41c, (1952) .

8.

J,R, Tata, L . Ernester and E . Suranyi, Biochem . Biophv . Acta . 6~0, 461, (1962) ,

(1957),