AN ANALYSIS OF THE POSITIVE INOTROPIC EFFECT OF ANAPHYLAXIS WITH POTASSIUM CONTRACTURE IN GUINEA PIG ATRIA, IN CONNECTION WITH THOSE OF INOTROPIC AGENTS

AN

ANALYSIS

OF

THE

ANAPHYLAXIS IN

WITH

GUINEA

PIG

UENO,

INOTROPIC

POTASSIUM

ATRIA,

THOSE KIRA

POSITIVE

OF

IN

OF

CONTRACTURE

CONNECTION

INOTROPIC

MASAMICHI

EFFECT

WITH

AGENTS

MATANO

ANDAYOSHIRO

NAKAZAWA

Departmentof Pharmacology,NagasakiUniversitySchoolof Medicine,Nagasaki Received for publication May 28, 1964

The anaphylactic

manifestation

is an increase in the contractile

in the atrium

a primary

manifestation

It was also found in our laboratory action

anaphylaxis sinoatrial

Furthermore,

in its height (4).

during

prolongation

anaphylaxis

of

action

atrial

records of action

potential

occurs as

muscle fibers (1).. of the trans

pig is prolonged of specialized

of action potential

has also been reported duration

during

fibers in the

showed the increased slope of the prepotential

Similar observation

potential

it has not been

that the reaction

taking place in cardiac fiber of guinea

pig

Although the reac

and adrenaline,

that the time course of repolarization

of proper

node during anaphylaxis

any changes segment

potential

(5, 6).

serotonin

(3) or DCI (4). So it is probable

effect of anaphylactic

membrane

from a sensitized guinea

tension and in the beat rate (1, 2).

tion is similar to the response of histamine, blocked by antihistaminics

isolated

without

change in intestinal

by Katsh and Marshall

(7). So the

in muscle fiber seems to be a ubiquitous

change occurring in the anaphylactic manifestation. On the other hand, some results have been presented indicating that the duration of the action potential is one factor determining the contractility: changes in temperature alter both electrical and mechanical activity (8,9), and tension and action potential duration decrease progressively as frequency increased in the rat atrium (9) and the guinea pig atrium (6), and several studies reported a change of comparable magnitude in action potential duration and contractility (10-13). These observations, however, do not imply that change in contractility must be associated with changes in action potential duration: that has also been confirmed in a number of studies (14-16). Therefore, the use of intracellular microelectrodetechnique to study electrical activity of cardiac muscle fibers has not provided much new information on the relationship between electrical and mechanical responses, and further investigation with different approaches is needed to discuss mechanism of the inotropic effect. In order to obtain further informations on the mechanism of anaphylactically induced inotropic action it seemed of interest to study the effect on contraction when muscle membrane is depolarized in the definite level. This can be done by studying 上野

昭 ・俣 野

正 道 ・中 沢

与 四郎

the participation induced

by

of most

drugs

tion

(17-19).

inotropic

of anaphylactic

application on the The

action

manifestation

of depolarizing potassium

authors in guinea

potassium

contracture

have

also

pig atria

on

chloride

have

studied on the

the

contractures

solution.

not been effects

potassium

of auricle

of

preparation

However,

reported

with

various

agents

attitudes

a few excep having

an

contracture.

METHODSAND MATERIALS About one hundred and fifty left atria taken from exsanguinated guinea pigs were used in the experiment. Immunological sensitization of the guinea pig was carried out by intravenous injection of anti-egg-albumin rabbit serum, having 1x 128 to 1x 256 antibody title (0.3ml per 100g body weight), 24 hours prior to the experiment. The left atria suspended in 32-33°C, 15 ml bath, having an aspirating orfice to keep a proper fluid level during replacements of solutions and sintered glass filter at the bottom to provide bubbling. mixture of 95% oxygen and 5% carbon dioxide. The tension was recorded by an unbonded strain gage transducer (Shinkoh Com munication Co., Ltd.: UL-120-10 or UL-120-50), in connection with either an automatic balancing strain recorder (AS-2) or a dynamic strain amplifier (DS6-P) and direct recording microammeter (Yokogawa Electric Works Co., Ltd.), with 600 mg in initial load. Arrangement of the apparatus was shown in Fig. 1. Bath solutions used in the experiment were Ringer's solution composed of NaC1 154 mm, KCI 5.6 mm, CaC12 2.2 mm, glucose 5.0 mm, NaHCO3 5.95 mm per liter, and

FIG.

1.

Arrangement

1, strain 4, recorder 8, muscle

gage

of

the

apparatus.

; 2, chromium

; 5, muscle holder

with

bath

plating

still

; 6, aspirating

stimulating

electrode

wire

; 3,

orfice

strain

amplifier

; 7, inflow

; 9, glass

filter.

tubing

; ;

a potassium

solution composed of 160 mm, glucose 5.0 mm, NaHCO3

The solution will be described The potassium

contracture

with the potassium tension development

of 400 ml of the solution

which provides

in the potassium

with Ringer's

5.95 mm per liter.

solution in this paper.

of the atrium was induced by replacing Ringer's

solution by perfusion

with an excess aspiration was replaced

as the potassium

solution,

to maintain

solution then

a proper

the developed

the bath

fluid level.

was completed,

solution

through

After

the potassium

tension

quickly

the

solution

disappeared,

and electric drive of the atrium was begun with rectangular pulses of 5 msec in duration, 120 pulses per minute at supramaximal voltages for a predetermined period of time. The stadium tracture.

of electrical

In order

driving

to obtain

in details in the following Observations

sulfate

period

contracture,

potential

throughout sulfate

potassium

precautions

con

described

microelectrode

are as follows : egg albumin

caffeine

BaC12.2H2O 0.532 mg/ml, CaC1, . 2H2O 0.32 mg/ml,

out in

described (5). Used agents and

histamine-HC1

40,ug/ml,

were carried

solution by intracellular

the experiment

0.01 ,ag/ml,

1.0 i g/ml, nicotine

further

of the atrium

is the same as that previously

adrenaline-HCl

following

were needed.

the exposure to the potassium

The method

its final concentrations 20,ug/ml,

a reproducible

chapter

of the transmembrane

some cases during technique.

is a reconditioning

1.0 pg/ml,

serotonin

with sodium

acetylcholine

benzoate

chloride

(Merck) creatinine 1.0 mg/ml,

0.01 ,ag/ml.

RESULTS

1. Normalfeature of the potassiumcontracture An extremely slow development of tension, a potassium contracture, was seen as the atrium was exposed to the potassium solution (5-10 minutes), after electrically driven in Ringer's solution for a relatively short :period of time (10-15 minutes). And the solution was replaced with Ringer's solution when the exposure period was terminated

FIG.

2 A.

Full

record

of

contractile

responses

of

guinea

pig

atrium

to the

potassium

solution. The denote obtained

record the

was

times between

proceeded of

exposure

8 and

from

right

to the

15. See text.

to

potassium

left.

The

solution,

numbers and

uniform

below

the

responses

record were

and

driven

electrically

for

15 minutes

(reconditioning next exposure

interval). And to the potassium

was repeated

in the

same

then the solution

manner,

and

so forth. In

this

of the

tension

exposure or

circumstance,

was first increased

was advanced,

10 times,

and

formly

several

shows

a course

in the this

steady

paper

otherwise

up

responded

of a typical state.

in

uni Fig. 2 B FIG. 2 B. Time course of a typical potassium contracture in a steady state. K. solution in, denote the bath solution was replaced by the potassium solution ; Ringer in, the potassium solution was replaced by Ringer's solution ; E.S. in, circuit of electrical stimulation was closed. (a), (b) and (c) show the time, drug administrations are carrying out. See text.

contracture

Results

concerned the

to 5

discussed

only

steady

with

state

in the

unless

stated.

Magnitude potential exposure

then

as the

usually

times (Fig. 2 A).

are

contracture

development

of

the

transmembrane

of atrial fibers to the potassium

during solution

the ap

peared as 11.4 mV (average of 6 cases) against -90 mV in Ringer's solution .

outer fluid, whereas

it appeared

as

2. Modificationof the potassiumcontracturedue to anaphylacticmanifestationand agentswhichchange contractileforce Application of antigen and inotropic agents were carried out in the following four manners: a) adding with the potassium solution, b) in the completed contracture state,

FIG.

3.

Enhancing

solution

on

effect the

of

contracture.

Ca

given

with

the

potassium

c) in reconditioning interval in which atria are driven electrically, d) adding with the potassium solution in Ca-depleted state (Figs. 2B, 6). a. Effectsof the specificantigen and agents applied with the potassiumsolution Developed tension of potassium contracture in sensitized atrium was slightly enhanced (10-20%) by the potassium solution containing antigen (egg albumin) in a concentration of 2 mg%. But no effect was observed in the normal atria by antigen, and by adre naline, histamine, serotonin, nicotine and acetylcholine (agents of group A) applied in this manner. While the magnitude of the contracture was enhanced 10-20% by BaC12, caffeine and CaCl2 (Fig. 3) (agents of group B). It is noticeable that caffeine and BaCl2 enhanced the contracture as well as CaC12 and that there is a distinct difference between the agents of group A and the B in their mode of action on the potassium contracture. b. Effectsof ' the agents in the depolarizedstate When caffeine was added in the completed potassium contracture state, a near completely depolarized state ( 11.4 mV against outer fluid), another increase of tension was obtained on the course of the potassium contracture (Fig. 4). And a similar effect was observed by adding of BaCl2 and CaCl2. But adrenaline, histamine, serotonin, nicotine and acetylcholine did not modify the normal course of the potassium contracture. Antigen application for sensitized atria also showed no effect.

FIG.

5.

Effect

ditioning FIG.

4.

An

increase

the

contracture

the

completed

of

tension

caused contracture

by

on

the

caffeine state.

course

of

given

in

of

acetylcholine

interval

Acetylcholine 3 minutes in

3.

prior

on was to

given

the added

the

in

recon

contracture. in

potassium

driven

state

exposure

c. Changes of the contracture when agents were given in the reconditioning interval The agents were added in two to five minutes prior to the exposure solution

in the reconditioning

interval,

in which atria are driven

and their influences on followed potassium The magnitude serotonin,

nicotine

of the contracture and acetylcholine

contracture

in Ringer's

solution,

were observed.

was slightly increased (Fig. 5).

of the potassium

by adrenaline,

And increased

histamine,

20-30% by CaCl2j caffeine

and BaCl2. A marked

enhancement

the administration It is significant they are applied been normally under

of the contracture

in sensitized atria

was also produced

by

of antigen. that every agent enhances

in the reconditioning activated

the magnitude

interval

in which

by electric stimulation.

the presence of the agents

of the contracture

the excitable

In other words, repetitive

of group A produce

a factor acting

after

membrane

has

excitations

inotropically.

d. Effects of the agents on the contracturein Ca depletedstate After the atrium soaked in Ca free Ringer's solution instead of the normal one in the reconditioning interval, it was exposed to the potassium solution several times. The tension development went down and became a state responding uniformly with small contracture (Fig. 6). The condition could be understood as Ca depleted state of the

FiG.

6.

Action

of

1, 2 and solution ing

atrium.

application contracture substitute

effect

atrium

acetylcholine

with

the

of CaCl2, as the

in role

was

interval

No further

to a sensitized

Ba

on

the

3, normal used ; at

potassium

potassium instead

of

state.

potassium

solution

the

normal

Ringer's

also

state.

This

depleted

the

in

indicates

solution.

with the potassium serotonin,

no effect

in the same

Ringer's

recondition

potassium

histamine,

of this state.

state.

4 Ca free

antigen

showed fact

Ca

solution

with

adrenaline,

caffeine

in the contracture

in ; since

by applying

And

BaCl2 (Fig. 6) and

of Ca

normal

12, Baa was administered

was observed

of this

contracture contracture

on the contracture,

manner that

solution

nicotine

Ba

produced and

and but

marked

caffeine

can

DISCUSSION The present the potassium applied

investigation

contracture

indicates

of guinea

with the depolarizing

Ringer's

solution,

contracture.

a period

And

application

with the potassium contructure,

potassium

solution

circumstances

potassium

serotonin,

caffeine,

enhance

administration,

the

potassium

which is especially

Above findings indicate tion is of somewhat

state has

marked

nature

during

anaphylaxis

is applied

with the potassium

It is apparent

concerned

modification

is demonstrated

depleted

on the potassium (agents of

without

regard

from that

of

state.

action of anaphylactic

of agents

of action

to the method manifesta

mentioned

above.

Further

duration

in atria

observed

role in anaphylactically

induced

potential

of the contracture

is observed when antigen

of anaphylactically

with action on contractile by addition

induced

mechanism

of antigen

force enhancing

effect is

of muscle fiber, since no

in the depolarized

state

and

Ca

state.

Contracture administrations contracture.

enhancing

The mechanism functioning

membrane

excitation

under

On the other hand, of methods

the presence

of application.

enhance

finding

of action

to affect the relation

between

of Ca in this way is already

pointed

tigation

gives also an evidence

caffeine

reproduce

is a probable contracture

connection

between

attitudes

and the rate of Ca-uptake

our laboratory

a role to enhance

depolarization

of action

that

act similarly

the

irre

mode of

mechanism

contraction.

Mode

(19). Present

inves

as Ca, since Ba and

in Ca depleted

of these agents

state.

There

in the potassium

pig atria, as previously

that the agents of group A and anaphylaxis

the

on contractile and

of

force enhan

contracture

indicates

out by Niedergerke

of the atrium

in guinea

induced

the potassium

clearly

that Ba and caffeine

the potassium contracture

of the

may be to alter a certain

plays

action of the agents of group B is to act directly

or alternatively

interval

that is to say, repeating

in anaphylactically

caffeine The

excitation,

of these agents

is also considered

only when their

reconditioning

action, therefore,

on membrane

Ca, Ba and

A is demonstrable

state, i.e. in the

of the inotropic

depending

force. Similar mechanism cement.

inotropic

effect of agents of group

are made in the driven

membrane

spective

namely

solution.

that mechanism

not directly

of antigen,

and acetylcholine

in the Ca depleted

because the enhancement

in

state), and application

no influence

(1, 5, 6) may not play a fundamental

force enhancement,

state

contracture only when they were added In contrast, agents of group B, Ca, Ba

that the mode of inotropic

different

in the driven

was

in the series of the potassium

nicotine

contracture

they also suggest that the prolongation

applied

state (depolarized

histamine,

in

only when antigen

in the administration

group A) increase magnitude of the potassium in the driven state prior to the contracture. and

and interval

contracture

solution in Ca depleted

while adrenaline,

effect of anaphylaxis

is recognizable

of the reconditioning

the other

in completed

that positive inotropic

pig atria

reported

increase Ca-uptake,

from and Ba

and caffeine decrease

it (1).

From above findings it is probable

that anaphylactically

effect differs from that of Ba or caffeine tion between

Ca and Ba or caffeine

induced positive inotropic

and that there may exist a substrate

in a certain

part

competi

of the cell membrane.

SUMMARY Anaphylactically was analyzed agents

induced

by its attitude

force enhancing against

which act inotropically

1. Potassium

effect in the isolated

potassium

in the guinea

contracture

contracture,

of the guinea pig atrium

And transmembrane

tion appeared 2. induced

as

11.4 mV against

by the administration

state prior

to the contracture,

state and Ca-depleted The enhancing

acetylcholine driven 4.

with

of the sensitized

the potassium antigen

the administration

effect of adrenaline,

state few minutes regard

solu

solution,

was applied

atrium

was

and

marked

in the

driven

in the completed

contracture

state had no effect.

on the contracture

Administrations

several

fiber in the potassium

of the contracture

was evoked when while

atrial

was reproduced

the outer fluid.

of antigen

in the contracture

with those

was induced by the replacement

The contracture

of the proper

A slight increase in magnitude

enhancement

3.

potential

and compared

pig atrium

pig atrium.

of Ringer's solution with 160 mm KCI solution. times.

guinea

histamine,

was demonstrated

serotonin,

nicotine

and

also of

only when the agent is added in the

prior to the contracture. of Ca, Ba and caffeine

tracture

without

marked

in the Ca depleted

to the

enhanced

the magnitude

method of the administration,

of the con

but this was especially

state. REFERENCES

1) UENO,A. : Proceedings of the 16th General Assembly of the Japan Medical Congress, Osaka, Vol. 1, 176 (1963) (Japanese) 2) NAKAZAWA, Y., SAKAI,O. ANDKOBATA, S.: MsshinIgaku 45, 147 (1958) (Japanese) 3) KIHARA,F. : NagasakiIgaku Z. 34, 1612 (1959) (Japanese) 4) KIDO,M. : Acta med.nagasaki9, 29 (1964) 5) UENO,A., MURAKAMi, K. ANDNAKAZAWA, Y. : Ibid.8, 41 (1964) 6) MATAYOSHI, M. : NagasakiIgaku Z. : 39, 234 (1964) (Japanese) 7) KATSH,S. ANDMARSHALL, J.M.: Amer.J. Physiol.196, 39 (1959) 8) UENO,A. : Unpublished data 9) 10) 11) 12) 13) 14) 15)

HOLLANDER, P.B. ANDLEYDEN,M.E.: CirculationRes. 3, 604 (1955) BURGEN, A.S.V. ANDTERROUX, K.G.: J. Physiol.119, 139 (1952) KLEINFELD, M., GREENE,H., STEIN,E. ANDMACIN,J.: Amer.J. Physiol.181, 35 (1955) KLEINFELD, M., STEIN,E. ANDMYERS,S. : CirculationRes.2, 488 (1954) WEBB,J., LEYDEN,M.E. ANDHOLLANDER, P.B. : Ibid. 4, 332 (1956) DUDDEL, J. ANDTRAUTWEIN, W.: Cardiologia25, 344 (1154) HOFFMAN, B.F. ANDBINDLER, E.: Amer.J. Physiol.185, 95 (1956)

16) TRAUTWEIN,W. AND DUDDEL,J.: Arch. ges. Physiol. 260, 24 (1954) 17) LUTTGAU,H. AND NIEDERGERKE,R.: J. Physiol. 143, 486 (1958) 18) OTSUKA, M. AND NONOMURA,Y.: J. Pharmacol. 141, 1 (1963) 19) NIEDERGERKE,R.: .J. Physiol. 134, 584. (1956)