THE RELATION OF ENDOGENOUS CATECHOLAMINE TO THE ACTION OF STROPHANTHIN-G ON ATRIAL CONTRACTIONS OF RABBITS AND GUINEA-PIGS

THE

RELATION

ACTION

OF

OF

ENDOGENOUS

STROPHANTHIN-G OF

RABBITS

CATECHOLAMINE ON

AND

ATRIAL

TO

THE

CONTRACTIONS

GUINEA-PIGS

JIRO SUGIMOTO AND NAOTAKE IIDA Department of Pharmacology, KansaiMedicalSchool,Moriguchi, Osaka Receivedfor publicationJanuary 21, 1965

It is generally accepted that the remarkable effect of digitalis on heart falure is caused by its specific action on the contractile mechanism of the heart muscle. The mode of pharmacological action of digitalis glycosides have been studied by many in vestigators, and it has been claimed that digitalis might have some influence on the energy production mechanism of the heart. The metabolic pathway of heart muscle contraction is not positively influenced by digitalis glycosides, though they do bring about an increase in contractile force [Wollenberger (1), Bing et al. (2, 3)]. Szent-Gyorgyi (4) reported that actomyosin is influenced by ATP and inorganic ions but not by digi talis. However, it has been reported that ATP-ase activity in the muscle cell membrane of rat hearts is promoted by digitalis in therapeutic dosage [Repke (5)]. On the other hand, Hadju and Szent-Gyorgyi (6) reported that the decrease in K-ion concentrations in the heart muscle induces an increase in contraction, and digitalis augments an efflux of the K-ions in the heart muscle. On contrary, Klaus et al. (7) reported that the influx of 42K-ions into the heart muscle cells was increased by digitalis in therapeutic doses. The cardiac action of digitalis has been explained by some investigators as that of an autonomic drug due to the result that digitalis increases adrenaline action [Donielpolu (8), Fujita (9)]. Our experiment were carried out to discover the relation of digitalis action to endo genous catecholamine mobilization in the atria from the standpoint that the catechol amines in heart muscle are mostly in a bound form and the contractile function of heart is controlled by the released catecholamine available. METHODS Rabbits weighing about 1.5 to 2.5 kg and guinea-pigs weighing about 200 to 350 g were used in the experiment. These animals were sacrified without anesthetic through hemorrage by cutting both common carotid arteries. Immediately thereafter, the heart was isolated and put into Locke's solution saturated with oxygen at 30° to 31°C. Locke's solution contained 9.0 g sodium chloride, 0.42 g potassium chloride, 0.24 g calcium chlo 杉本

治 良 ・飯 田

尚武

ride, 0.2g sodium

bicarbonate

prepared from the heart, means of a spring lever.

and 1.0 g dextrose

the atrial

was begun

contractions

The concentrations (Merk), reserpine

(Serpasil,

atria

were

or more.

contractions

at a constant were recorded

level.

hydrochloride,

L-methionine

CIBA) and strophanthin-G

to where

The changes in ampli

after the addition

of the drugs were expressed in teams of g/ml.

the following : adrenaline

The

about one hour after setting the preparation

could be maintained

tude and rate of the atrial

of water.

and the contractions were recorded on a smoked drum by The thus prepared atria continued to contract rhythmically

in Locke's solution at 30' to 31V for 15 hours The experiment

per litre

(Sumitomo

of the drugs.

The drugs used were Chemical),

nicotine

(Takeda).

RESULTS

1) The effectof strophanthin-Gon atrial contraction In concentrations lower than 1.5x 10-6, strophanthin-G increased the amplitude of atrial contraction one to two minutes after addition to a degree of 20 to 50% of the original level, but only slightly increased the rate of contraction in rabbit atria. After addition of the intermediate concentrations of from 1.5x10-' to 1.75x10-', the positive inotropic effect of strophanthin-G continued for 20 minutes to about one hour, and then the rhythmical contraction gradually changed to arrhythmia, but the atrial move ment continued for an hour or more. In concentrations higher than 1.75x 10-6, the drug quickly induced the arrhythmia after a positive inotropic effect continued for 5 to 20 minutes, but these arrhythmic contractions gradually decreased in amplitude until they ceased within about one hour after the addition. 2) The effectof adrenalineafter additionof strophanthin-G The augmented rhythmic contraction following the addition of strophanthin-G in lower concentrations responded to an additional application of adrenaline (10-8 to 10-6), but the response of the atria to adrenaline in the presence of strophanthin-G was similar to that in the absence of the same drug. The arrhythmical contractions induced by strophanthin-G in intermediate concentrations were abruptly stopped by the additional application of adrenaline 10-6. In the higher concentrations of strophanthin-G, the ar rhythmical contractions were gradually decreased in amplitude, and stopped after a transient augmentation in amplitude by the addition of adrenaline 10-6. 3) The effectof strophanthin-Gafter additionof adrenaline The contractions of the atria augmented by additions of adrenaline 10-8 to 10-7, responded to an additonal application of strophanthin-G 10-6, but the response to the latter drug in the presence of adrenaline was similar to the one observed in its absence. 4) The effectof nicotineafter additionof' strophanthin-G Nicotine 10-5 first decreased contraction of guinea-pig atria both in amplitude and rate for 10 to 30 seconds and then increased it to a degree even surpassing the original. When strophanthin-G 10 had been added 3 to 5 minutes before nicotine 10-5, the later

FIG. 1. Isolated guinea-pig atria ; responses to nicotine (10-5) were augmented 'by preceding addition of strophanthin -G (S-G 10-6). Numerals above the record give atrial frequencies (beats/min). At (W) the bath fluid was changed. The concentration of each drug is shown in g/ml. Time inter vals are one minute.

stimulant 5)

action

of nicotine

was strengthened

as shown in Fig. 1.

The effect of strophanthin-G after addition of L-methionine

L-Methionine in concentrations 10-6 to 10-5 did not affect the contraction of atria, but in concentrations of 10-4 to 10-3 after a latency of about 3 to 4 minutes L-methionine mildly and

gradually

augmented

the atrial

contractions

slightly in rate (10). In the presence of L-methionine of strophanthin-G

was augmented

20 to 30% in amplitude 10-4 the positive

inotropic

as shown in Fig. 2.

FIG. 2. Isolated guinea-pig atria ; responses to strophanthin-G (S-G 10-6) were augmented by preceding addition of L-methionine (10-4). Numerals above the record give atrial frequencies (beats/min). At (W) the bath fluid was changed. The concentration of each drug is shown in g/ml. Time inter vals are one minute.

and effect

6)

The effect of reserpine and the resumption rf atrial contraction by strophanthin-G

The atrial contractions of rabbits were arrested when reserpine to the bath. If the arrested atria were moreover kept in reserpine solution for 60 to 90 minutes,

these arrested

when the bath fluid was repeatedly G 10-6 caused the resumption had

changed.

of atrial

been excised from animals

experiment,

a concentration

as described

did not resume

spontaneously,

But a small concentration

movement

pretreated

were arrested by reserpine

by strophanthin-G

atria

3 x 10-5 was added containing Locke's

(Fig. 3).

with reserpine

of strophanthin

When rabbit

atria, which

1 mg/kg 24 hours

3 x 10-5, the resumption

even

before the

of atrial movement

above was not seen even after the application

caused

of as large

as 2 x 10

FIG. 3. Isolated rabbit atria ; the contraction was arrested by the addition of reserpine (3 x 10-5) and thereafter atria were kept in reserpine containing Locke's solution for about one hour. At (W) the bath fluid was changed, but resumption of atrial contraction did not occur spontaneously. Thus treated atria could be resumed by strophanthin-G (10-6). Numerals above the record give atrial frequencies (beats/min). The concentration of each drug is shown in g/ml. Time intervals are one minute.

7)

The effect of strophanthin-G on atria excised from rabbit pretreated with reserpine subcutaneously 24 hours before experiment As mentioned

isolated

atrial

experiment

in experiment

contraction

atria,

these atria

but

continued

adrenaline

pretreated

which had

Strophanthin-G could.

even after added

tion of a small amount

The

of adrenaline

uptake

G 10-6 was added to the bath containing inotropic amount tion than

effect.

animals

been pretreated

did not contract

contractions

into the atrial

resumed tissue.

uptake,

in the case of normal

required atria.

larger

doses of re

in Locke's solution by

was washed out, probably

these atria,

to arrest

in dose of 1 mg/kg. with larger

beating added

of

adre

by the utiliza

When strophanthin

strophanthin-G

As shown in Fig. 4, the atria which continued

of adrenaline

able

was also observed in the

could not resume the arrested

atrial

adrenaline

of strophanthin-G

same result

1.2 to 1.5 mg/kg 24 hours before experiment,

after setting the preparation. naline

The

with atria excised from reserpine

In some cases the rabbit serpine

1, the concentration

was 1.75 x 10-6.

showed a positive

to contract

doses of strophanthin-G

by a small

to stop contrac

FIG. 4. In this case the atria, excised from the rabbit pretreated with reserpine 1.5 mg/kg subcutaneously 24 hours before experiment, did not contract in Locke's solution spontaneously and also after the addition of strophanthin-G (S-G 10-s). The atria could be resumed by adrenaline (Adr 10-5) and contracted even after the added adrenaline was removed. Strophanthin-G showed a positive inotropic effect on the atria, which were finally stopped by two times or more doses of strophanthin-G than that in the case of normal atria. Numerals above the record give atrial frequencies (beats/min). At (W) the bath fluid was changed. Time intervals are 10 minutes.

DISCUSSION The positive studied and

inotropic

in relation

and

toxic

to the available

effects

of strophanthin-G

endogenous

on atrial

contraction

in atrial

tissues

catecholamine

were

of rabbits

guinea-pigs. In experiments

cholamine

and

But,

increased Nicotine

and

in the presence

of exogenous

showed

at the

nerve

caused

by the

release

ending

structure

As reported

available effect

To

of endogenous catecholamine

structure

adrenergic

by Sugimoto

fib°rs.

bound

released

of

the

by the

acute

or

10'

relation effect

animals

and

the latter

may

and

also

subacute

to augment

a

the atrial

the

the positive

of L-methionine

release

of

inotropic

in experiment

metabolism. and

had a lesser amount of reserpine.

later has

of nicotine.

accelerating that

be

nerve

the

strophanthin-G

application

to catecholamine

action

in the

strengthened that

of strophanthin-G

effect

of acetyl

is considered

which

bath. depressive release

cells

The fact

was

by the

fibers

to 10-3 by

in the presence

the

we used

to the

As strophanthin-G

has some

catecholamine,

be caused

chromaffin

catecholamine.

between

of .strophanthin-G

added

4, it is assumed

was augmented

strophanthin-G

contraction

effect

i.e. an initial

cholinergic

the

et al. (11), L-methionine

from

the relation

after

in

may

cate

on atrial

2, the toxic

movement,

of the

concentrations

the effect of exogenous

catecholamine

former

catecholamine

high

1 and

in experiment

endogenous

catecholamine

that

The

between

of strophanthin-G

atrial

of noradrenaline

of strophanthin-G

clarify

effect.

of nicotine

in the

5, is proof

effect on

ending

of the

effect

contractions

a diphasic

stimulatory

to the

effect

in experiments

choline

relation

inotropic

interaction

as shown

subsequent

stimulant

3, a mutual

the positive

not observed. was

2 and

The

the

mobilization of endogenous

opinions

on

the

difference

between the effects of strophanthin-G

among many investigators.

and reserpinized

Tanz (12) recognized the dependence

effect of strophanthin-G

on catecholamine

Yelnosky

Morrow

et al. (13), and

on normal

muscle was independent

content

of the positive inotropic

in the papillary

et al. (14) reported

of reserpine

that

pretreatment.

hearts differ

muscle of cats, but

the contractile

force of heart

On the other hand,

Cairoli et al.

(15) mentioned that the response of papillary muscle of cats -to strophanthin-G was decreased but not abolished by reserpine pretreatment. In our experiments, the interesting effects of strophanthin-G were observed by study ing the response

of reserpine

pretreated

rabbit

et al. (17) and Tachi et al. (18) reported

atria

to this drug.

that the atria

arrested

Tachi (16), Matsuo

by the addition

of reser

pine were resumed by the application of adrenaline, n•oradrenaline and dopamine, after the added reserpine had been washed out. Moreover, Sugimoto et al. (11) described that the resumption Catron.

of the reserpine

In experiment

arrested

6, as the atria

atria

was also induced

arrested

by reserpine

by

L-methionine

could be resumed

or

by ap

plication of strophanthin-G, it may be assumed that the action of some amounts of endogenous catecholamine which were too small to maintain the rhythmic contraction of the atria was accelerated by strophanthin-G. If the atria excised from the rabbits had been pretreated with reserpine 1 mg/kg subcutaneously, no resumption could be ob served after endogenous

strophanthin-G catecholamine

to accelerate

the arrested

dose of strophanthin-G

application. after reserpine atria

In the latter treatment

atria

even after its release by strophanthin-G.

to manifest

a positive

inotropic

cholamine

it is assumed

is necessary

effect on the atria, certain amount, storage amount

continued

to contract

take into the atrial case of normal moderate

by utilization

develops

different

But, as shown of a extremely

Therefore,

on the toxic

catecholamine

cate

of the the dose

from that of atria

small amount that

than a

independently

in experiment

it may be assumed

the toxic effect of strophanthin-G

From

catecholamine

excised

7, the atria

which

of catecholamine

tissue, were stopped by more doses of strophanthin-G

atria.

which endogenous

animals.

of endogenous

to reveal its positive inotropic

more endogenous

atria was hardly

animals.

doses does not influence

cases, although

contain

atria did not

As for the toxic effect of strophanthin-G,

of normal

pretreated

of strophanthin-G

effect of strophanthin-G

of catecholamine.

to stop the contraction from reserpine

if the atria

the inotropic

pretreated

that the presence of some amounts

for the action

and

decreased

However, the

effect on normal

differ from the one seen in atria excised from the reserpine these two results,

the extremely

may be assumed to be insufficient

reserpine

than

up in the

pretreatment

effect of strophanthin-G

in ordinary

may decrease in the case of atria

has been extremely

in in

exhaused.

SUMMARY The

positive inotropic

and

toxic effects of strophanthin-G

were studied on atrial

preparations from rabbits and guinea-pigs. Strophanthin-G augmented the action of available endogenous catecholamine

released

by nicotine,

and

L-methionine The

the positive

inotropic

which may accelerate

positive inotropic

exogenous adrenaline. the application

the metabolism

effect of strophanthin-G

The rabbit

of strophanthin-G

same resumption

effect of strophanthin-G

atria arrested after

did not occur with atria

of endogenous on atria

by reserpine

the reserpine

by the pretreatment

with larger

.

was not synergistic 3x10-'

excised from animals

does

catecholamine

by

were resumed

with by

had been washed

pine 1 mg/kg 24 hours before the experiment. The atria, in which the endogenous catecholamine decreased

was strengthened

may

of reserpine,

out , while the pretreated with reser

have

did not

been

extremely

respond

to stro

phanthin-G but could respond to the same drug in the presence of endo as well as exogenous catecholamine. In such cases of extremly exhausted endogenous catechol amine content,

however,

the toxic effect of strophanthin-G

The toxic effects of strophanthin-G those on atria

on normal

atria

was remarkably

which were excised from rabbits or guinea-pigs

1 mg/kg 24 hours before experiment,

decreased . from

were scarcely different pretreated

though they were potentiated

with reserpine

by excessive exogenous

catecholamine. REFERENCES 1) WOLLENBERGER, A. : Pharmacol.Rev.1, 311 (1949) 2) BING,R.J., CHOUDHURY, J.D., MICHAL,G. ANDKAKO,K.: Ann. intern. Med. 49, 1201 (1958) 3) BING,R.J. ANDDANFORTH, W.H. : J. Amer.med. Ass. 172, 438 (1960) 4) SZENT-GYORGYI, A.: ChemicalPhysiologyof Body and Heart Muscle,Academic Press Inc., New York (1953) 5) REPKE,K. : Proc. 1st Int. Pharmacol.Meet. Vol. 3, p. 68, Pergamon Press, Oxford, London, New York, Paris (1963) 6) HADJU,S. ANDSZENT-GYORGYI, A. : Amer.J. Physiol.168, 171 (1952) 7) KLAUS,W., KUSHINSKY, G. ANDLULLMANN, H.: Proc. 1st Int. Pharmacol.Meet. Vol. 3, p. 211, 8) 9) 10) 11)

Pergamon Press, Oxford, London, New York, Paris (1963) DANIELPOLU, D. : Cardiologia12, 66 (1947) FUJITA,S. : Folia pharmacol.japon. 46, 27 (1950) (Japanese) TAMINO,K.: J. KansaiMed. Sch. 15, 63 (1933) (Japanese) SUGIMOTO, J., TAMINO,K., KADOKAWA, C., IIDA, N. AND MORITA,M. : THIS JOURNAL14, 150

(1954) 12) TANZ,R.D.: Pharmacologist 2, 95 (1960) 13) YELNOSKY, J.H. ANDERVIN,R. : Amer.Heart J. 62, 687 (1961) 14) MORROW, D.H., GAFFNEY, T.E. AND BRAUNWALD, E. : J. Pharmacol.140, 236 (1963) 15) CAIROLI,V., REILLY,J. ANDROBERTS, J. : Fed. Proc. 20, 122 (1961) 16) TACHI,S. : Folia pharmacol.japon. 53 449 (1962) (Japanese) 17) MATSUO,T. ANDTACHI,S. : THISJOURNAL12, 191 (1962) 18) TACHI,S., MATSUO,T., FUJIWARA, M. ANDTODA,N. : Ibid. 12, 197 (1962)