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RECOVERING EFFECT OF ACETYLCHOLINE ON THE DEPRESSED ATRIAL TRANSMEMBRANE POTENTIAL OF THE RESERPINIZED RABBIT BY THE ADRENOLYTICS
RECOVERING
EFFECT
DEPRESSED
ATRIAL OF
THE
OF
ACETYLCHOLINE
TRANSMEMBRANE
RESERPINIZED THE
RABBIT
ON
THE
POTENTIAL BY
ADRENOLYTICS
YOSHIMI
MISU
Departmentof Pharmacology,Facultyof Medicine,KyotoUniversity,Sakyo-ku,Kyoto Received for publication August 8, 1963
In the previous paper (1) the effects of the adrenolytics on the transmembrane poten tial of the extirpated rabbit's heart were reported. The adrenolytics exerted depressive effects on the action potential, i.e. decrease in the rate, prolongation of the de- and re polarization phases and depression of the amplitude with subsequent complete abolition of the atrial non-pacemaker potential. The resting potential was slightly decreased. However, the pacemaker potential, though it was slightly or moderately depressed, con tinued to fire rhythmically. In this condition addition of adrenaline or noradrenaline in the concentration which exerted a positive inotropic effect on the intact heart re started the potential rhythm. However, the recovery of the transmembrane potential was transient and incomplete even by addition of higher concentration of either amine. Physiological role of noradrenaline in the heart (2-5) on the spontaneous automati city of the atrium has not been hitherto fully elucidated. Though the effects of the adrenolytics on the transmembrane potential of the heart may relate with the adrenergic mechanism, Matsuo et al. (6) have shown the increase of noradrenaline in the rabbit's heart in response to the intravenous injection of dibenamine (10 mg/kg) but not to the same procedure of yohimbine (5 mg/kg) or chlorpromazine (5 mg/kg). The biphasic responses of the heart to acetylcholine have been observed by several authors (7-14). The restarting effect of acetylcholine on the atrial preparation in which rhythmical contraction had been abolished by quinidine, eserine and paludrine (12, 13) and by the lowering of environmental temperature (14) has been reported. The present investigation has been attempted to elucidate the mechanism of the standstill of the atrial transmembrane potential induced by the adrenolytics by study ing the restarting and recovering effects of adrenaline, noradrenaline and acetylcholine in the isolated atrium of rabbit which had been pretreated with reserpine. It has been also expected to explain the mode of action of the endogenous catecholamine and acetylcholine on the atrial non-pacemaker potential. 三須 良実
METHODS Th f; Methods and procedures same as those described
in the previous
were used in the experiments. toneally
employed
in the present paper
The animals
(1).
experiments
About
thirty
were almost the
reserpinized
received 0.5 mg/kg of reserpine
at 48 and 24 hours before the extirpation
rabbits intraperi
of heart, of which doses were enough
to deprive the heart catecholamine almost completely (15, 16). The recordings of the transmembrane potential were made at least one hour after the fixation of the prepara tion on the cork plate in the modified Tyrode solution, maintained at 30°C and saturated with 95% 02 and 5% CO2. The effects of the adrenolytics on the atrial non-pacemaker potentials of the reserpinized rabbits were divided into four stages according to the previous results, described as R-stage I, II, III and IV. RESULTS I. The rate and nized rabbits Table
configuration
differed
of the atrial
considerably
1, the reserpinized TABLE
heart 1.
Effect
* : Standard
resting
of the
potential
error
amplitude,
reserpinization
of
the
on atria
animal.
bradycardia.
the in
of the reCerpi As shown
Though
in
no notable
transmembrane rabbits.
time of the action
the 90% repolarization of the reserpinized
of the repolarization
II.
isolated
the depolarization
was observed,
phase of the repolarization, and Fig. 5-A).
a significant
of
potentials
of the normal
.
this level of the action potential The prolongation
transmembrane
from those
exhibited
potentials
difference
Effects of Reserpinization
time and
and
the
the total duration
potential
at
atrium were significantly
prolonged.
time consisted of those of the phase 1, the early
and of the phase 3, the terminal
phase (Fig. 3-A, Fig. 4-A
Effects of Dibenamine on the Reserpinized Atrium
Fig. 1 shows the effects of 5x10-'
of dibenamine
on the rate of the atrial
action
potentials of the reserpinized rabbit and the untreated one. The initial and transient increase of the rate was more marked and longer-lasting in the reserpinized atrium than in the untreated
one.
About forty minutes after the application
of dibenamine
the
FiG. 1. Effects of dibenamine, yohimbine, and chlorpromazine on the spontaneous rate of isolated atria in untreated and reserpinized rabbits. Each point in the figure represents the mean value in 4 to 6 animals. increased
rate
about
twenty
thmic
action
maker
area
time
for
turned
thereafter,
and
the rhy
potentials
in the
non-pace
disappeared
totally.
The
the
disappearance
action
potential
in the
reserpinized
treated
to a . decrease
minutes
was
of the
significantly
atrium
lag
atrial shorter
than
in the un
one.
The effects of 5 x 14-5 of dibenamine the configuration
of the
atrial
on
non-pace
maker potential are shown in Fig. 2-A to -c and Fig . 5-A to -D. Five to fifteen mi nutes after the application the slight prolongation FiG. 2. Effects of dibenamine on the trans membrane potentials of the atrial fibers in the reserpinized rabbit (A-C). The recovery course after the addition of noradrenaline (NAd) is shown in D-F. A : Before. B : 15 min after the ap plication of dibenamine (5 x 10-5). C : 50 min after. D : 1 min after the ad dition of NAd (10-s). E : 3 min . after washing out. At 10 min before wash ing-out 5 x 10_6of NAd was added. F : 4 min after the addition of NAd (5 x 10-6). Time calibration shows intervals of 100 msec. Vertical bar is a voltage calibration of 100 mV (See Text).
tion time and rounding were observed
of dibenamine
of the depolariza of the overshoot
(Fig. 2-B).
tion of the phase
The prolonga
1, 2 and
3 of the re
polarization and the depolarization phase resulted in the increase of the total dura tion of the The
action
potential
slight depression
the further overshoot
rounding
(R-stage
of the
amplitude,
of the upstroke
as well as the
I).
more
and
marked
prolongation of the depolarization time were observed twenty to thirty minutes
after the application marked
of dibenamine,
prolongation
of the repolarization
of the slope of phase 3 increased the stage II of the atrial dibenamine marked
prolongation
potential
obtained
about
of the untreated
In this stage the rate of the potential
of the configuration
of the transmembrane
are shown in Fig. 5-C. depressed,
The amplitude
minutes turned
potential
hand, in
produced by
though
the similarly
observed after
(1).
The R
the application
to decrease.
was markedly
effect was more marked
of
The changes
associated with the decrease
of the action potential
and the prolonging
rabbit
prolonged,
to forty
retardation
On the other
time was definitely
thirty
The concomitant
with the further
profoundly.
time was not markedly
of the depolarization
stage III was usually
gressively
time associated
the total duration
transmembrane
the repolarization
dibenamine.
as shown in Fig. 5-B (R-stage II).
of rate and pro
in the repolarization
phase than in the depolarization phase. The prolongation of the former phase, especially that of the phase 3, resulted in more marked prolongation of the total duration. The resting
potential
was little affected.
the depolarization heart
than in the untreated
to twenty action
minutes.
potential
The
manifestation
of the
inflection
phase in the R-stage II to III was less frequent one (Fig. 5-B and -C).
Thereafer,
gradually
the action potential
the further
to a miniature
disappeared
about
or notch in
in the reserpinized
The R-stage III lasted usually ten
depression
of the amplitude
transferred
the
wave, as shown in Fig. 2-C (R-stage IV), and
sixty minutes
after the application
of dibenamine
(Fig. 5-D). The resting potential was slightly decreased (Fig. 2-C and 5-D). The repeated washing of the atrium in which the action potential was abolished by dibenamine
did not restart
or noradrenaline reported
depressed
application
by dibenamine
of dibenamine,
transmembrane
potential
of the repolarization
rhythm,
to the similarly
the application either amine added.
treated
atria
found
between
additions of either recovered
On the other hand,
depressed
reserpinized (Fig. 2-E and
transient
produced
of adrenaline
(Fig. 2-D).
As was
amine to the untreated to the level before the the configuration
of the
with the full recovery
the repetitive
atrium
of the configuration
of dibenamine
rate-decreasing
rhythm
the rate transiently
while the same procedure
time (1).
was usually
III. The
recovered
while the addition
of the potential
to the level of the stage II accompanied
of the rate and the recovery
minutes
potential
in the previous paper, the repetitive
atrium
amine
the
evoked the reappearance
additions
of either
the complete
recovery
to the level of the R-stage I or before
-F).
even if higher
However, concentration
the recovering of either
effect of amine
was
Effects of Chlorpromazine on. the Reserpinized Atrium effect of 10-5 of chlorpromazine
is shown in Fig. 1. both preparations.
after the application
No significant
difference
on both reserpinized of the decrease
As shown in Fig. 3-A to -E, about
of 10-5 of chlorpromazine
the amplitude
and un
of rate was ten to fifteen of the
action
potential of the reserpinized atrium was depressed as well as that of the untreated one (R-stage I). About ten to twenty minutes thereafter, the amplitude of the action poten
tial was more markedly increase
depressed and the
of the total duration,
sisted of the marked repolarization
which
prolongation
time
and
the
longation
of the depolarization
observed
(R-stage
II).
of the repolarization
The
con of the
slight
pro
time, was prolongation
phase resulted mainly
from that of the phase 3 (Fig. 3-C), while this was not observed, but the prolongation .of the depolarization
phase was markedly
observed in the stage II of the untreated atrium (1). The appearance of the two-step wise notches in the depolarization easily observed FIG. 3. Effects of transmembrane fibers in the The recovery renaline (Ad) A : Before.
chlorpromazine on the potentials of the atrial reserpinized rabbit (A-E). after the addition of ad is shown in F. B : 10 min after the ap
miniature
waves (Fig. 3-E).
the stage when the action The reserpinized application
potential
of chlorpromazine
was rarely seen in the reserpinized
atrium.
membrane minutes
potential after
about
the application
in Fig. 3-D (R-stage
III).
tion of the repolarization more marked
are shown
The
prolonga
phase was also
than that of the depolariza
tion phase in this stage. action potentials seventy-five
of trans
fifty to sixty
The
disappeared
minutes
depressed
totally about
after the application
of chlorpromazine without manifesting the the resting potential was slightly decreased in
However,
atrium
atrium,
The changes of the configuration
plication of chlorpromazine (10-5). C : 40 min after. D : 60 min after. E : 70 min after. F : 3 min after the addition of Ad (10-6). Time calibration shows intervals of 100 msec. Vertical bar is a voltage calibration of 100 mV (See Text).
phase,
in the untreated
disappeared
(Fig. 3-D and -E).
in which the action was not restarted
potential
had been abolished
by the repetitive
washing
by the
of the pre
paration, but was restarted by the addition of adrenaline or noradrenaline. Then, the configuration of the transmembrane potential was recovered to the level of the poten tial in the R-stage I.
The recovery took place most early in duration
tion phase and latest in that of the depolarization covery effect of either amine potential
occurred
unless the addition IV.
Fig. 1 illustrates action potential
transient
of either
of the repolariza However,
and the reabolition
the re
of the action
amine was repeated.
Effects of Yohimbine on the Reserpinized Atrium
the time course
was abolished
bine in the untreated bine decreased
was usually
phase (Fig. 3-F).
atrium.
of the
rate-decreasing
effect of yohimbine.
about two hours after the application On the
the rate gradually
The
of 10-5 of yohim
other hand , the same concentration of yohim potential disappeared totally about
and the action
eighty minutes after the application reserpinized
in the
atrium.
Fig. 4-A to -D shows the time course of the effects of 10-5 of yohimbine on the con figuration of the non-pacemaker potentials in the reserpinized atrium. Five to ten minutes
after
the application
of the companied
overshoot
the round
and
upstroke
ac
with the slight prolongation
of
the de and repolarization
phases were ob
served (R-stage
prolongation
I).
The
the de and repolarization that
of the
depression
of
times, especially
phase 3 associated
of the amplitude
with
the
was observed
fifteen to twenty minutes after the applica tion (R-stage nounced
II in Fig. 4-B).
prolongation
tion phase, especially resulted
in the
the total C). the
of the
pro
repolariza
that of the phase 3,
marked
duration
The
prolongation
(R-stage
FIG. 4. Effects of yohimbine on the trans membrane potentials of the atrial fibers in the reserpinized rabbit (A-D). The recovery course after the addition of ace tylcholine (ACh) is shown in E-H. A : Before. B : 24 min after the appli cation of yohimbine (10-5). C : 45 min after. D : 70 min after. E : 2 min after the addition of ACh (10-6). F : 4 min after washing out. G : 5 min after the second addition of ACh (10-6). H : 70 min after the second washing-out. At 10 min before the washing-out 10-6 of ACh was added. Time calibration shows intervals of 100 msec. Vertical bar is a voltage calibra tion of 100 mV (See Text).
of
III in Fig. 4
The notch, which appeared often in depolarization phase of the action
potential in this stage of the untreated atrium, appeared less frequently in the re serpinized atrium (Fig. 4-B and -C). The R-stage III was obtained about thirty to fifty minutes after the application ed for twenty after,
the
to thirty
shortening
minutes.
of the de
and last There and
re
polarization times which might had been brought about by the extreme depression of the amplitude
was observed, and the action
minutes after the application tial was decreased The washing, level of the phase effect
action but
of the
potential
disappeared
totally about eighty
(R-stage IV in Fig. 4-D).
The resting poten
slightly in the R-stage III and IV (Fig. 4-C and -D).
potential
abolished
was by the rate
of yohimbine
before
transmembrane
addition the
by
of adrenaline
application
potential
yohimbine
in the
usually preceded to that of the of either amine was also transient.
was
restarted
or noradrenaline
of yohimbine R-stage
not
I.
depolarization
and The
and
to the recovery
one.
level of
However,
by the
repetitive
recovered
to the
of configuration the
repolarization the
recovering
V. Restarting and Recovering Effects of Acetylcholine The addition pacemaker
of 10'
potentials
to 10-6 of acetylcholine
were abolished by the application
yohimbine restarted the action potential. from that of adrenaline or noradrenaline acetylcholine
at the optimal
tion recovered
to the untreated
of dibenamine,
in which non
chlorpromazine
or
The restarting effect of acetylcholine differed in the following points : 1) The addition of
concentration
the configuration
atrium
and
the repetition
of washing
of the depressed transmembrane
the prepara
potential
almost com
pletely, i.e. to the level before the application of adrenolytics. 2) However, the recover ing effect of acetylcholine on the rate of the potential was incomplete. 3) The recover ing effect of acetylcholine
on the configuration
longer-lasting
effects of acetylcholine
(17).
These
of the transmembrane were observed
treated atrium but also in the reserpinized one. Fig. 4 shows the depressing effect of 10-5 of yohimbine action
potential
after
action
potential
was restarted
acetylcholine from 10'
the addition
(Fig. 4-E).
of acetylcholine
about one to three atrium
potentials in response to higher
were usually
few in number
repetitive
washing
gradually
was long-lasting
and progressively
(Fig. 4-H).
the decreased
(Fig. 4-G).
However,
in the
The
of 10-6 of
one.
of acetylcholine
The re
above 10-5 atria
which
could be restarted
in all cases.
and the declined
of the
was in the range
The reserpinized
of acetylcholine
of acetylcholine
of acetylcholine, potential
of acetylcholine
concentration
was
atrium.
after the addition
less lasting.
to the single addition
of the action
increased
and therefore
and the addition
minutes after the addition amplitude
in the reserpinized
as well as in the untreated
started action
had not responded
and the reactivation
minutes
The effective concentration
to 10-6 in the reserpinized
potential
not only in the un
Within
resting potential,
by the
three
to five
the depressed
slope of the depolarization
Thereafter,
phase
this effect of acetylcholine
reserpinized
atrium,
the prolonged
re
polarization phase, especially phase 3 was not fully recovered to the level of that before the addition of the adrenolytics even by the repetitive washing and addition of acetyl choline
(Fig. 4-G and -H).
Fig. 5-E to -P shows the time course of the recovery of the depressed non-pacemaker potential produced by the application of 5x10-' atrium, in responses to acetylcholine, noradrenaline The action potential was restarted
was added
of the atrium which had been abolished
one and half minutes after the first addition
5-E) and the peak The nutrient
of dibenamine to the reserpinized and the combination of both drugs.
effect was observed
solution containing twice between
after the third addition and depolarization
three
dibenamine
Fig. 5-F and -G.
and
half
was washed
by 5 x 10-5 of dibenamine
of 10-6 of acetylcholine minutes, out and
thereafter
time of the action potential
The resting potential, recovered
completely
(Fig. 5-F).
10-6 of acetylcholine
Fig. 5-G shows the potential
of 10-6 of acetylcholine.
(Fig.
three
minutes
the amplitude
to the initial level,
while the prolonged repolarization time, especially the phase 3 was not by these pro cedures. This stage continued for forty-five minutes after the third addition of acetyl chline
(Fig. 5-H).
The action potential
recorded
thirteen
minutes
after
the fourth
ad
FIG. 5. Effects of dibenamine on the transmembrane potentials of the atrial fibers in the reserpinized rabbit (A-D). The recovery course after the additions of acetylcholine (ACh) and noradrenaline (NAd) is shown in E-P. A : Before. B : 35 min after the application of dibenamine (5 x 10-~). C : 39 min after. D : 41 min after. E : 1.5 min after the first addition of ACh (10-6). F : 5 min after. Between F and G ; 3 min after F ; a : washed out the nutrient solution. 10 min after a; b : the second addition of ACh (10-6). 27 min after b; c : the third addition of ACh (10-6). G : 3 min after c. H : 45 min after. I : 13 min after the fourth ad dition of ACh (10-6). J : 3 min after the addition of high dose of ACh (10-5). K : 30 min after. L : 5 min after the simultaneous additions of ACh (10-6) and NAd (10-'). M : 3 min after the addition of NAd (10-6). N : 8 min after. 0 : 12 min after. P : 1 min after the simultaneous additions of ACh (10-6) and NAd (10-6). Time calibration shows intervals of 100 msec. Vertical bar is a voltage calibration of 100 mV (See Text).
dition of 10-6 of acetylcholine did not show the recovery of the repolarization phase, especially phase 3 (Fig. 5-I). The addition of 10-5 of acetylcholine shortened markedly the repolarization time (Fig. 5-J), but the effect was transient (Fig. 5-K). The action potential observed five minutes after the combined addition of 10-6 of acetylcholine and l0' of noradrenaline (Fig. 5-L) shows the pronounced shortening of the phase 3 in the repolarization phase followed with the slight hyperpolarization of ~phase 4. This ten dency of hyperpolarization became more apparent by the further addition of 10-6 of noradrenaline (Fig. 5-M, -N, and -0), and the repolarization time was also shortened, as shown in Fig. 5-P. The next combined addition of 10-6 of acetylcholine and nor adrenaline produced a full recovery of the resting potential, the amplitude and the de and repolarization times of the action potential in the reserpinized atrium to the initial level.
DISCUSSION In the present bine
experiment,
on the non-pacemaker
serpinized
rabbits
adrenaline
and
the effects
of dibenamine,
transmembrane
potentials
as well as the restarting acetylcholine
on the
and
atrial
chlorpromazine
of the isolated
recovering
potentials
effects
depressed
and
atria
yohim
in the
re
of adrenaline,
nor
by these adrenolytics
were studied. The
isolated atrium
of the
the rate and a prolongation non-pacemaker
area.
reserpinized
rabbit
in the 90% repolarization
of heart
changes
of the heart
noradrenaline,
demonstrated
the action
that
potential
1) there is a linear
correlation
of the total duration
at the frequency
the slight decrease
of the rate
derive from the depletion was more marked by Muskus chronotropic releasing cluded
in the reserpinized
(15) and response
Crout
that
the rate-increasing
in the reserpinized
the
still of the atrium reserpinization
was shortened
on the atrial
The
atrium
of the
ministration
of chlorpromazine renaline,
which
The
Their
effects
against
positive
probably
acts 'by
result
it is con
directly
with the
The rate-decreasing effects and yohimbine were more , The
potentiating
indicate
lowered
results also support of the presence the opposing
effects of
that the endogenous the intravenous
elevated the level of noradrenaline
are independent
the
the lag time needed for the stared
to the adrenolytics
though their results does not exclude
noradrenaline
It has been shown
does not relate
of 5 mg/kg of yohimbine
did not affect it.
effects of the adrenolytics
from
but may
of dibenamine
abolishes
to tyramine,
by the reserpinization.
of 10 mg/kg of dibenamine the same procedure
prolonga
heart,
to 5 x 10'
presence of noradrenaline opposes these effects of the adrenolytics. Matsuo et al. (6) in this laboratory have shown that though of rabbit,
of the phase
marked
From the above-mentioned
and consequently,
responses
of
of the phase 2
may not derive
contraction
reserpinization
effect of dibenamine
atrium,
total duration
and 3) the duration
presence of the endogenous noradrenaline in the atrium. of dibenamine as well as the same effects of chlorpromazine marked
et al. (19)
noradrenaline.
heart
noradrenaline.
the
and Zaimis (18),
the
atrium than in the normal one.
et al. (16) that
from
the histological
from that
of the rate in response
of the guinea-pig's
the endogenous
mainly
in reserpinized
of the spontaneous
increase
in
from sixty to three hundred/
below fifty/min.
of the endogenous
The initial and transient
between
frequency derives
of stimulation
tion of the phase 3 _of the repolarization
potential
muscle of dog's heart, Hoffman
and not from that of the phase 3 of the repolarization, 2 is constant
in
effects may derive
as were shown by Withrington
Using the papillar
and the rate of stimulation
min, 2) the shortening
These
decrease
of the phase 1 and
shown by many authors (2-5), though
by reserpinization,
should also be considered. have
time of the action
The latter effect consisted of the prolongation
3, and not of the phase 2 of the repolarization. depletion
showed a considerable
and
that
ad
in the atria of 5 mg/kg
the conclusion
of the endogenous
that the norad
effects of the endogenous
the effects of the adrenolytics.
of adrenolytics
on the configuration
of the spontaneous
atrial
trans
membrane
potential
were studied comparatively
As has been shown in the previous lytics on the normal resulted
atrium
report,
in the untreated
was the prolongation
in the increase of the total duration,
depolarization reserpinized frequent
phase (1). atrium
appearance
of the notch.
of the depolarization
the application
a less prolongation However,
atria.
effect of the adreno
and the appearance
On the other hand,
produced
and reserpinized
the most pronounced
time which
of the notch in the
of the adrenolytics
of the depolarization
to the
time and a less
the increase of the total duration,
which
mainly derived from the prolongation of the phase 3 of repolarization, was observed. The prolongation of the phase 3 developed usually associated with the decrease of the rate, but it was also observed to 5 x 10-5 of dibenamine. rization
Since the reserpinization
phase of the atrial
adrenaline markedly
lessens the in the
even in the stage of initial transmembrane
prolongation
normal
atrium,
increase of the rate in response
of the animal
potential,
the depletion
of the depolarization
and augments
the
prolongs
time,
prolongation
the repola
of the atrial
which
nor
was observed
of the repolarization
by the adrenolytics. It may be concluded that the presence of the endogenous norad renaline in the atrium maintains the normal length of the repolarization time and op poses to the prolonging effect of the adrenolytics on the repolarization time. The reserpinized atrium in which spontaneous action potential had been abolished by the adrenolytics
was not restarted
was easily restarted
by the addition
atrium
almost
the repetitive rization
the same events have
by the washing-out of adrenaline
of the nutrient
or noradrenaline.
been observed.
However,
additions of either amine and washing-out depolarization
In the normal normal
atrium
recovered the prolonged
in the
repola
time to almost initial level, especially in the case of dibenamine,
of the prolonged
solution but
time, the depressed amplitude
but the recovery
and the decreased
resting
potential did not progress beyond the level in the stage II of the effects of the adreno lytics (1). On the other hand, the prolonged repolarization time of the reserpinized atrium caused by the adrenolytics recovery
was fully recovered
of the depolarization
ing potential
level associated
of the action potential
to the exogenous
atrial noradrenaline
the reserpinization
catecholamine.
and the sensitivity
No linear
of cat decreased correlation
with the
and the rest
to the level in the R-stage I of the effects of the adrenolytics.
and Zaimis (18) showed that heart
to the normal
time and amplitude
Withrington
the sensitivity
between
of the
the content
of the atria to exogenous noradrenaline
of
in guinea
pig was shown by Crout et al. (16). The hypersensitivity of the pressor response of the reserpinized cat to catecholamine was demonstrated by Burn et al. (20). The more ef fective reversibility of adrenaline or noradrenaline against the depressing effects of the adrenolytics on rate and configuration of the transmembrane potential in reserpinized atria coincides with the hypersensitivity of the reserpinized structure to either amine. The recovery of the depressed potential in the reserpinized atria by adrenaline or nor adrenaline was usually transient and the restarted action potential disappeared again at the variable time-length as well as in the untreated ones. The addition of l0' to 10-6of acetylcholine restarted the untreated (17) and the
reserpinized atria in which the spontaneous action potentials had been abolished by the adrenolytics. The addition of acetylcholine produced a full recovery of the resting potential, the amplitude and depolarization time of the action potential to the initial level. However, the prolonged repolarization time of the reserpinized atrium and the rate of the potential were not recovered to the normal level . Further, the recovering effect of acetylcholine was longer-lasting with the exception of the repolarization phase . The prolongation of the repolarization phase which had been effective to acetylcholine alone was restored effectively to the normal level by the combined addition of acetyl choline and noradrenaline. The addition of noradrenaline to the reserpinized atrium which had been recovered by acetylcholine as described above induced sometimes a sign of the hyperpolarization in the phase preceded with the repolarization one . It has been postulated that the heart responds to acetylcholine biphasically (7-14). The stimulating effect of acetylcholine has been believe to derive from the direct effect on the ganglion cell or chromaffin cell within the heart (7, 8) or from the release of adrenaline-like substance from the structure (9-11). The restarting and recovering effects of acetylcholine against the depressive action of the adrenolytics on rate and configuration of the atrial transmembrane potential in reserpinized rabbit show clearly the cholinergic mechanism of acetylcholine itself . The important role of the synthesis and liberation of the endogenous acetylcholine in the heart for initiation and maintenance of the spontaneous beat has been shown by Burn et al. (12, 13, 21, 22). They hypothe sized that the critical level of the endogenous acetylcholine in the heart was a deter mining factor for the initiation of the contractile stimulus and the addition of acetyl choline exhibited an inhibitory or excitatory effect according to the rate of synthesis of the endogenous acetylcholine. The restarting effect of acetylcholine on the reserpi nized atrium of which the action potential had been abolished by the adrenolytics might have been elucidated in association with the increase of the resting potential by acetyl choline or vagal stimulation, as reported by Burgen and Terroux (23), Hutter and Trautwein (24), and Hoffman and Suckling (25). From the results described above, it is assumed that the exogenous and probably endogenous acetylcholine regulates and maintains the depolarization phase of the atrial action potential, while adrenaline or noradrenaline does the repolarization phase . The less prolongation of the depolarization time produced by the adrenolytics in the reser pinized atrium than in the normal one is assumed to derive from the change of the rate of the content of the endogenous noradrenaline to acetylcholine, i.e. the overwhelming majority of the endogenous acetylcholine, by which the effect of the adrenolytics on the depolarization time is opposed. The results presented by Malhotra et al. (26) that the intravenous injection of 0.5 mg/kg of reserpine in dogs increased the extractable acetylcholine by 75.3% in the S-A node, 48.3% in the right atrium and 44.4% in the right ventricle support this assumption. In the unpublished experiment the author confirmed that the untreated rabbit's atrium in which the action potentials had been abolished by the application of a moderately high concentration of DO was not re
started by adrenaline or noradrenaline, but restarted by the addition of acetylcholine or the repetitive washing. However, the prolongation of the repolarization time pro duced by DCI was not recovered to the initial level even by these procedures. The results also support this assumption. The relation between the effects of adrenolytics and acetylcholine on the atrial potentials" will be discussed in details in the succeeding report. SUMMARY Effects of 5 x l0-' of dibenamine, 10-' of chlorpromazine and yohimbine were studied on the non-pacemaker potentials of the isolated atria of the rabbit pretreated with reserpine. Competitive effects of adrenaline, noradrenaline and acetylcholine against these adrenolytics were compared between reserpinized and untreated atria. 1. The action potentials in the isolated atrium of the reserpinized rabbit showed the considerable decrease in rate and the prolongation of the 90% repolarization time, especially the phase 3 of the repolarization. 2. The initial rate-increasing effect of dibenamine, and the rate-decreasing effect of dibenamine, chlorpromazine and yohimbine were manifested more markedly in the reserpinized atrium than in the untreated one. The lag time needed for the standstill of the atrium was shortened by the reserpinization. 3. The application of the adrenolytics produced less prolongation of the depola rization time and less frequent appearance of the notch on the depolarization phase in the reserpinized atrium than in the untreated one. More marked prolongation of re polarization phase, especially the phase 3, was observed in the reserpinized preparation. 4. The reserpinized atrium in which the spontaneous action potentials had been abolished by the adrenolytics failed to restarted by washing-out, but was easily restarted by adrenaline or noradrenaline as well as the untreated one. However, the repetitions of the addition either amine and washing-out recovered the depressed transmembrane potential more effectively in the reserpinized atrium than in the untreated one. Espe cially, the prolonged repolarization time of the reserpinized atrium was fully recovered to the normal level. However, the recovery by adrenaline or noradrenaline was also incomplete and transient in the reserpinized atria. The action potentials in the reser pinized atria disappeared again at the variable time-length after the addition as well as in the untreated one. 5. The addition of acetylcholine restarted the action potential which had been abolished by the adreno]ytics in the reserpinized atria. The decreased resting potential, the depressed amplitude and prolonged depolarization time of the action potential were completely recovered to initial level, but the prolonged repolarization time and decreased rate of the action potential failed to recover. The recovering effect of acetylcholine was longer lasting than that of adrenaline or noradrenaline. 6. The prolongation of the repolarization phase was effectively restored to the normal level by the combined addition of acetylcholine and noradrenaline. Occasionally,
the addition
of noradrenaline
produced
a hyperpolarization
at the phase following
the
repolar,ization. 7. regulates atrium,
From the results described and maintains while
above, it is assumed
the depolarization
noradrenaline
as a possibility
that acetylcholine
phase of the action potential
does the repolarization
in the rabbit's
one.
REFERENCES 1) MISU,Y. : THISJOURNAL13, 167 (1963) 2) BERTLER, A., CARLSSON, A, ANDROSENGREN, E. : Naturwissenschaften 43, 521 (1956) 3) PAASONEN, M.K. ANDKRAYER,O.: J. Pharmacol.123, 153 (1958) 4) HAGEN,P. : Pharmacol.Rev. 11, 361 (1959) 5) MATSUO,T.:THIS JOURNAL12, 62 (1962) 6) MATSUO,T., HATTORI,K. ANDHOJO, T.: To be published 7) :ROTHBERGER, G J ANDSACHS,A. : Cardiology2, 71 (1938) 8) SPADOLINI, I..AND DOMINI,G.: Arch. Fisiol. 40, 147 (1940) 9) HOFFMAN,F., HOFFMAN, E.J., MIDALETON, S. ANDTALESNIK, J. : Amer.J. Physiol.144, 189 (1945) 10) McDoWALL., J.R.S.: J. Physiol.104, 342 (1946) 1) MCNAMARA, B., KROP,S. ANDMcKAY,1E.A.: J. Pharmacol.92, 153 (1948) 12) BRISCOE, S. ANDBURN,J.H.: Brit. J. Pharmacol.9, 42 (1954) 13) BURN,J.H. ANDVANE,J.R.: J. Physiol.108, 104 (1949) 14) MARSHALL, J.M. ANDVAUGHAN MILLIAMS, E.M. Ibid. 131, 186 (1956) 15) MUSKUS, A.J. : J. Pharmacol.138, 296 (1962) 16) GROUT,J.R., MUSKUS,A.J. ANDTRENDELENBURG, U. : Brit. J. Pharmacol.18, 600 (1962) 17) MISU,Y. ANDTAKAORI, S. : To be published 18) WITHRINGTON, P. ANDZAIMIS,E. : Brit. J. Pharmacol.17, 380 (1961) 19) HOFFMAN,B.F. ANDSUCKLING, E.E. : Amer.J. Physiol.179, 123 (1954) 20) 21) 22) 23) 24) 25) 26)
BURN,J.H. ANDRAND,M.J.: J. Physiol.144, 314 (1958) BULBRING, E. ANDBURN,J.H.: Ibid. 108, 508 (1949) BURN,J.H. : Pharmacol.Rev. 6, 107 (1954) BURGEN, A.S.V. ANDTERROUX,K.G. : J. Physiol.120, 449 (1953) HUTTER,O.F. ANDTRAUTWEIN, W.: Nature,Lond. 176, 512 (1955) HOFFMAN, B.F. ANDSUCKLING, E.E.: Amer.J. Physiol.173, 312 (1953) MALHOTRA, C.L. ANDDAs, P.K. : Brit. J. Pharmacol.18, 190 (1962)
EFFECT
DEPRESSED
ATRIAL OF
THE
OF
ACETYLCHOLINE
TRANSMEMBRANE
RESERPINIZED THE
RABBIT
ON
THE
POTENTIAL BY
ADRENOLYTICS
YOSHIMI
MISU
Departmentof Pharmacology,Facultyof Medicine,KyotoUniversity,Sakyo-ku,Kyoto Received for publication August 8, 1963
In the previous paper (1) the effects of the adrenolytics on the transmembrane poten tial of the extirpated rabbit's heart were reported. The adrenolytics exerted depressive effects on the action potential, i.e. decrease in the rate, prolongation of the de- and re polarization phases and depression of the amplitude with subsequent complete abolition of the atrial non-pacemaker potential. The resting potential was slightly decreased. However, the pacemaker potential, though it was slightly or moderately depressed, con tinued to fire rhythmically. In this condition addition of adrenaline or noradrenaline in the concentration which exerted a positive inotropic effect on the intact heart re started the potential rhythm. However, the recovery of the transmembrane potential was transient and incomplete even by addition of higher concentration of either amine. Physiological role of noradrenaline in the heart (2-5) on the spontaneous automati city of the atrium has not been hitherto fully elucidated. Though the effects of the adrenolytics on the transmembrane potential of the heart may relate with the adrenergic mechanism, Matsuo et al. (6) have shown the increase of noradrenaline in the rabbit's heart in response to the intravenous injection of dibenamine (10 mg/kg) but not to the same procedure of yohimbine (5 mg/kg) or chlorpromazine (5 mg/kg). The biphasic responses of the heart to acetylcholine have been observed by several authors (7-14). The restarting effect of acetylcholine on the atrial preparation in which rhythmical contraction had been abolished by quinidine, eserine and paludrine (12, 13) and by the lowering of environmental temperature (14) has been reported. The present investigation has been attempted to elucidate the mechanism of the standstill of the atrial transmembrane potential induced by the adrenolytics by study ing the restarting and recovering effects of adrenaline, noradrenaline and acetylcholine in the isolated atrium of rabbit which had been pretreated with reserpine. It has been also expected to explain the mode of action of the endogenous catecholamine and acetylcholine on the atrial non-pacemaker potential. 三須 良実
METHODS Th f; Methods and procedures same as those described
in the previous
were used in the experiments. toneally
employed
in the present paper
The animals
(1).
experiments
About
thirty
were almost the
reserpinized
received 0.5 mg/kg of reserpine
at 48 and 24 hours before the extirpation
rabbits intraperi
of heart, of which doses were enough
to deprive the heart catecholamine almost completely (15, 16). The recordings of the transmembrane potential were made at least one hour after the fixation of the prepara tion on the cork plate in the modified Tyrode solution, maintained at 30°C and saturated with 95% 02 and 5% CO2. The effects of the adrenolytics on the atrial non-pacemaker potentials of the reserpinized rabbits were divided into four stages according to the previous results, described as R-stage I, II, III and IV. RESULTS I. The rate and nized rabbits Table
configuration
differed
of the atrial
considerably
1, the reserpinized TABLE
heart 1.
Effect
* : Standard
resting
of the
potential
error
amplitude,
reserpinization
of
the
on atria
animal.
bradycardia.
the in
of the reCerpi As shown
Though
in
no notable
transmembrane rabbits.
time of the action
the 90% repolarization of the reserpinized
of the repolarization
II.
isolated
the depolarization
was observed,
phase of the repolarization, and Fig. 5-A).
a significant
of
potentials
of the normal
.
this level of the action potential The prolongation
transmembrane
from those
exhibited
potentials
difference
Effects of Reserpinization
time and
and
the
the total duration
potential
at
atrium were significantly
prolonged.
time consisted of those of the phase 1, the early
and of the phase 3, the terminal
phase (Fig. 3-A, Fig. 4-A
Effects of Dibenamine on the Reserpinized Atrium
Fig. 1 shows the effects of 5x10-'
of dibenamine
on the rate of the atrial
action
potentials of the reserpinized rabbit and the untreated one. The initial and transient increase of the rate was more marked and longer-lasting in the reserpinized atrium than in the untreated
one.
About forty minutes after the application
of dibenamine
the
FiG. 1. Effects of dibenamine, yohimbine, and chlorpromazine on the spontaneous rate of isolated atria in untreated and reserpinized rabbits. Each point in the figure represents the mean value in 4 to 6 animals. increased
rate
about
twenty
thmic
action
maker
area
time
for
turned
thereafter,
and
the rhy
potentials
in the
non-pace
disappeared
totally.
The
the
disappearance
action
potential
in the
reserpinized
treated
to a . decrease
minutes
was
of the
significantly
atrium
lag
atrial shorter
than
in the un
one.
The effects of 5 x 14-5 of dibenamine the configuration
of the
atrial
on
non-pace
maker potential are shown in Fig. 2-A to -c and Fig . 5-A to -D. Five to fifteen mi nutes after the application the slight prolongation FiG. 2. Effects of dibenamine on the trans membrane potentials of the atrial fibers in the reserpinized rabbit (A-C). The recovery course after the addition of noradrenaline (NAd) is shown in D-F. A : Before. B : 15 min after the ap plication of dibenamine (5 x 10-5). C : 50 min after. D : 1 min after the ad dition of NAd (10-s). E : 3 min . after washing out. At 10 min before wash ing-out 5 x 10_6of NAd was added. F : 4 min after the addition of NAd (5 x 10-6). Time calibration shows intervals of 100 msec. Vertical bar is a voltage calibration of 100 mV (See Text).
tion time and rounding were observed
of dibenamine
of the depolariza of the overshoot
(Fig. 2-B).
tion of the phase
The prolonga
1, 2 and
3 of the re
polarization and the depolarization phase resulted in the increase of the total dura tion of the The
action
potential
slight depression
the further overshoot
rounding
(R-stage
of the
amplitude,
of the upstroke
as well as the
I).
more
and
marked
prolongation of the depolarization time were observed twenty to thirty minutes
after the application marked
of dibenamine,
prolongation
of the repolarization
of the slope of phase 3 increased the stage II of the atrial dibenamine marked
prolongation
potential
obtained
about
of the untreated
In this stage the rate of the potential
of the configuration
of the transmembrane
are shown in Fig. 5-C. depressed,
The amplitude
minutes turned
potential
hand, in
produced by
though
the similarly
observed after
(1).
The R
the application
to decrease.
was markedly
effect was more marked
of
The changes
associated with the decrease
of the action potential
and the prolonging
rabbit
prolonged,
to forty
retardation
On the other
time was definitely
thirty
The concomitant
with the further
profoundly.
time was not markedly
of the depolarization
stage III was usually
gressively
time associated
the total duration
transmembrane
the repolarization
dibenamine.
as shown in Fig. 5-B (R-stage II).
of rate and pro
in the repolarization
phase than in the depolarization phase. The prolongation of the former phase, especially that of the phase 3, resulted in more marked prolongation of the total duration. The resting
potential
was little affected.
the depolarization heart
than in the untreated
to twenty action
minutes.
potential
The
manifestation
of the
inflection
phase in the R-stage II to III was less frequent one (Fig. 5-B and -C).
Thereafer,
gradually
the action potential
the further
to a miniature
disappeared
about
or notch in
in the reserpinized
The R-stage III lasted usually ten
depression
of the amplitude
transferred
the
wave, as shown in Fig. 2-C (R-stage IV), and
sixty minutes
after the application
of dibenamine
(Fig. 5-D). The resting potential was slightly decreased (Fig. 2-C and 5-D). The repeated washing of the atrium in which the action potential was abolished by dibenamine
did not restart
or noradrenaline reported
depressed
application
by dibenamine
of dibenamine,
transmembrane
potential
of the repolarization
rhythm,
to the similarly
the application either amine added.
treated
atria
found
between
additions of either recovered
On the other hand,
depressed
reserpinized (Fig. 2-E and
transient
produced
of adrenaline
(Fig. 2-D).
As was
amine to the untreated to the level before the the configuration
of the
with the full recovery
the repetitive
atrium
of the configuration
of dibenamine
rate-decreasing
rhythm
the rate transiently
while the same procedure
time (1).
was usually
III. The
recovered
while the addition
of the potential
to the level of the stage II accompanied
of the rate and the recovery
minutes
potential
in the previous paper, the repetitive
atrium
amine
the
evoked the reappearance
additions
of either
the complete
recovery
to the level of the R-stage I or before
-F).
even if higher
However, concentration
the recovering of either
effect of amine
was
Effects of Chlorpromazine on. the Reserpinized Atrium effect of 10-5 of chlorpromazine
is shown in Fig. 1. both preparations.
after the application
No significant
difference
on both reserpinized of the decrease
As shown in Fig. 3-A to -E, about
of 10-5 of chlorpromazine
the amplitude
and un
of rate was ten to fifteen of the
action
potential of the reserpinized atrium was depressed as well as that of the untreated one (R-stage I). About ten to twenty minutes thereafter, the amplitude of the action poten
tial was more markedly increase
depressed and the
of the total duration,
sisted of the marked repolarization
which
prolongation
time
and
the
longation
of the depolarization
observed
(R-stage
II).
of the repolarization
The
con of the
slight
pro
time, was prolongation
phase resulted mainly
from that of the phase 3 (Fig. 3-C), while this was not observed, but the prolongation .of the depolarization
phase was markedly
observed in the stage II of the untreated atrium (1). The appearance of the two-step wise notches in the depolarization easily observed FIG. 3. Effects of transmembrane fibers in the The recovery renaline (Ad) A : Before.
chlorpromazine on the potentials of the atrial reserpinized rabbit (A-E). after the addition of ad is shown in F. B : 10 min after the ap
miniature
waves (Fig. 3-E).
the stage when the action The reserpinized application
potential
of chlorpromazine
was rarely seen in the reserpinized
atrium.
membrane minutes
potential after
about
the application
in Fig. 3-D (R-stage
III).
tion of the repolarization more marked
are shown
The
prolonga
phase was also
than that of the depolariza
tion phase in this stage. action potentials seventy-five
of trans
fifty to sixty
The
disappeared
minutes
depressed
totally about
after the application
of chlorpromazine without manifesting the the resting potential was slightly decreased in
However,
atrium
atrium,
The changes of the configuration
plication of chlorpromazine (10-5). C : 40 min after. D : 60 min after. E : 70 min after. F : 3 min after the addition of Ad (10-6). Time calibration shows intervals of 100 msec. Vertical bar is a voltage calibration of 100 mV (See Text).
phase,
in the untreated
disappeared
(Fig. 3-D and -E).
in which the action was not restarted
potential
had been abolished
by the repetitive
washing
by the
of the pre
paration, but was restarted by the addition of adrenaline or noradrenaline. Then, the configuration of the transmembrane potential was recovered to the level of the poten tial in the R-stage I.
The recovery took place most early in duration
tion phase and latest in that of the depolarization covery effect of either amine potential
occurred
unless the addition IV.
Fig. 1 illustrates action potential
transient
of either
of the repolariza However,
and the reabolition
the re
of the action
amine was repeated.
Effects of Yohimbine on the Reserpinized Atrium
the time course
was abolished
bine in the untreated bine decreased
was usually
phase (Fig. 3-F).
atrium.
of the
rate-decreasing
effect of yohimbine.
about two hours after the application On the
the rate gradually
The
of 10-5 of yohim
other hand , the same concentration of yohim potential disappeared totally about
and the action
eighty minutes after the application reserpinized
in the
atrium.
Fig. 4-A to -D shows the time course of the effects of 10-5 of yohimbine on the con figuration of the non-pacemaker potentials in the reserpinized atrium. Five to ten minutes
after
the application
of the companied
overshoot
the round
and
upstroke
ac
with the slight prolongation
of
the de and repolarization
phases were ob
served (R-stage
prolongation
I).
The
the de and repolarization that
of the
depression
of
times, especially
phase 3 associated
of the amplitude
with
the
was observed
fifteen to twenty minutes after the applica tion (R-stage nounced
II in Fig. 4-B).
prolongation
tion phase, especially resulted
in the
the total C). the
of the
pro
repolariza
that of the phase 3,
marked
duration
The
prolongation
(R-stage
FIG. 4. Effects of yohimbine on the trans membrane potentials of the atrial fibers in the reserpinized rabbit (A-D). The recovery course after the addition of ace tylcholine (ACh) is shown in E-H. A : Before. B : 24 min after the appli cation of yohimbine (10-5). C : 45 min after. D : 70 min after. E : 2 min after the addition of ACh (10-6). F : 4 min after washing out. G : 5 min after the second addition of ACh (10-6). H : 70 min after the second washing-out. At 10 min before the washing-out 10-6 of ACh was added. Time calibration shows intervals of 100 msec. Vertical bar is a voltage calibra tion of 100 mV (See Text).
of
III in Fig. 4
The notch, which appeared often in depolarization phase of the action
potential in this stage of the untreated atrium, appeared less frequently in the re serpinized atrium (Fig. 4-B and -C). The R-stage III was obtained about thirty to fifty minutes after the application ed for twenty after,
the
to thirty
shortening
minutes.
of the de
and last There and
re
polarization times which might had been brought about by the extreme depression of the amplitude
was observed, and the action
minutes after the application tial was decreased The washing, level of the phase effect
action but
of the
potential
disappeared
totally about eighty
(R-stage IV in Fig. 4-D).
The resting poten
slightly in the R-stage III and IV (Fig. 4-C and -D).
potential
abolished
was by the rate
of yohimbine
before
transmembrane
addition the
by
of adrenaline
application
potential
yohimbine
in the
usually preceded to that of the of either amine was also transient.
was
restarted
or noradrenaline
of yohimbine R-stage
not
I.
depolarization
and The
and
to the recovery
one.
level of
However,
by the
repetitive
recovered
to the
of configuration the
repolarization the
recovering
V. Restarting and Recovering Effects of Acetylcholine The addition pacemaker
of 10'
potentials
to 10-6 of acetylcholine
were abolished by the application
yohimbine restarted the action potential. from that of adrenaline or noradrenaline acetylcholine
at the optimal
tion recovered
to the untreated
of dibenamine,
in which non
chlorpromazine
or
The restarting effect of acetylcholine differed in the following points : 1) The addition of
concentration
the configuration
atrium
and
the repetition
of washing
of the depressed transmembrane
the prepara
potential
almost com
pletely, i.e. to the level before the application of adrenolytics. 2) However, the recover ing effect of acetylcholine on the rate of the potential was incomplete. 3) The recover ing effect of acetylcholine
on the configuration
longer-lasting
effects of acetylcholine
(17).
These
of the transmembrane were observed
treated atrium but also in the reserpinized one. Fig. 4 shows the depressing effect of 10-5 of yohimbine action
potential
after
action
potential
was restarted
acetylcholine from 10'
the addition
(Fig. 4-E).
of acetylcholine
about one to three atrium
potentials in response to higher
were usually
few in number
repetitive
washing
gradually
was long-lasting
and progressively
(Fig. 4-H).
the decreased
(Fig. 4-G).
However,
in the
The
of 10-6 of
one.
of acetylcholine
The re
above 10-5 atria
which
could be restarted
in all cases.
and the declined
of the
was in the range
The reserpinized
of acetylcholine
of acetylcholine
of acetylcholine, potential
of acetylcholine
concentration
was
atrium.
after the addition
less lasting.
to the single addition
of the action
increased
and therefore
and the addition
minutes after the addition amplitude
in the reserpinized
as well as in the untreated
started action
had not responded
and the reactivation
minutes
The effective concentration
to 10-6 in the reserpinized
potential
not only in the un
Within
resting potential,
by the
three
to five
the depressed
slope of the depolarization
Thereafter,
phase
this effect of acetylcholine
reserpinized
atrium,
the prolonged
re
polarization phase, especially phase 3 was not fully recovered to the level of that before the addition of the adrenolytics even by the repetitive washing and addition of acetyl choline
(Fig. 4-G and -H).
Fig. 5-E to -P shows the time course of the recovery of the depressed non-pacemaker potential produced by the application of 5x10-' atrium, in responses to acetylcholine, noradrenaline The action potential was restarted
was added
of the atrium which had been abolished
one and half minutes after the first addition
5-E) and the peak The nutrient
of dibenamine to the reserpinized and the combination of both drugs.
effect was observed
solution containing twice between
after the third addition and depolarization
three
dibenamine
Fig. 5-F and -G.
and
half
was washed
by 5 x 10-5 of dibenamine
of 10-6 of acetylcholine minutes, out and
thereafter
time of the action potential
The resting potential, recovered
completely
(Fig. 5-F).
10-6 of acetylcholine
Fig. 5-G shows the potential
of 10-6 of acetylcholine.
(Fig.
three
minutes
the amplitude
to the initial level,
while the prolonged repolarization time, especially the phase 3 was not by these pro cedures. This stage continued for forty-five minutes after the third addition of acetyl chline
(Fig. 5-H).
The action potential
recorded
thirteen
minutes
after
the fourth
ad
FIG. 5. Effects of dibenamine on the transmembrane potentials of the atrial fibers in the reserpinized rabbit (A-D). The recovery course after the additions of acetylcholine (ACh) and noradrenaline (NAd) is shown in E-P. A : Before. B : 35 min after the application of dibenamine (5 x 10-~). C : 39 min after. D : 41 min after. E : 1.5 min after the first addition of ACh (10-6). F : 5 min after. Between F and G ; 3 min after F ; a : washed out the nutrient solution. 10 min after a; b : the second addition of ACh (10-6). 27 min after b; c : the third addition of ACh (10-6). G : 3 min after c. H : 45 min after. I : 13 min after the fourth ad dition of ACh (10-6). J : 3 min after the addition of high dose of ACh (10-5). K : 30 min after. L : 5 min after the simultaneous additions of ACh (10-6) and NAd (10-'). M : 3 min after the addition of NAd (10-6). N : 8 min after. 0 : 12 min after. P : 1 min after the simultaneous additions of ACh (10-6) and NAd (10-6). Time calibration shows intervals of 100 msec. Vertical bar is a voltage calibration of 100 mV (See Text).
dition of 10-6 of acetylcholine did not show the recovery of the repolarization phase, especially phase 3 (Fig. 5-I). The addition of 10-5 of acetylcholine shortened markedly the repolarization time (Fig. 5-J), but the effect was transient (Fig. 5-K). The action potential observed five minutes after the combined addition of 10-6 of acetylcholine and l0' of noradrenaline (Fig. 5-L) shows the pronounced shortening of the phase 3 in the repolarization phase followed with the slight hyperpolarization of ~phase 4. This ten dency of hyperpolarization became more apparent by the further addition of 10-6 of noradrenaline (Fig. 5-M, -N, and -0), and the repolarization time was also shortened, as shown in Fig. 5-P. The next combined addition of 10-6 of acetylcholine and nor adrenaline produced a full recovery of the resting potential, the amplitude and the de and repolarization times of the action potential in the reserpinized atrium to the initial level.
DISCUSSION In the present bine
experiment,
on the non-pacemaker
serpinized
rabbits
adrenaline
and
the effects
of dibenamine,
transmembrane
potentials
as well as the restarting acetylcholine
on the
and
atrial
chlorpromazine
of the isolated
recovering
potentials
effects
depressed
and
atria
yohim
in the
re
of adrenaline,
nor
by these adrenolytics
were studied. The
isolated atrium
of the
the rate and a prolongation non-pacemaker
area.
reserpinized
rabbit
in the 90% repolarization
of heart
changes
of the heart
noradrenaline,
demonstrated
the action
that
potential
1) there is a linear
correlation
of the total duration
at the frequency
the slight decrease
of the rate
derive from the depletion was more marked by Muskus chronotropic releasing cluded
in the reserpinized
(15) and response
Crout
that
the rate-increasing
in the reserpinized
the
still of the atrium reserpinization
was shortened
on the atrial
The
atrium
of the
ministration
of chlorpromazine renaline,
which
The
Their
effects
against
positive
probably
acts 'by
result
it is con
directly
with the
The rate-decreasing effects and yohimbine were more , The
potentiating
indicate
lowered
results also support of the presence the opposing
effects of
that the endogenous the intravenous
elevated the level of noradrenaline
are independent
the
the lag time needed for the stared
to the adrenolytics
though their results does not exclude
noradrenaline
It has been shown
does not relate
of 5 mg/kg of yohimbine
did not affect it.
effects of the adrenolytics
from
but may
of dibenamine
abolishes
to tyramine,
by the reserpinization.
of 10 mg/kg of dibenamine the same procedure
prolonga
heart,
to 5 x 10'
presence of noradrenaline opposes these effects of the adrenolytics. Matsuo et al. (6) in this laboratory have shown that though of rabbit,
of the phase
marked
From the above-mentioned
and consequently,
responses
of
of the phase 2
may not derive
contraction
reserpinization
effect of dibenamine
atrium,
total duration
and 3) the duration
presence of the endogenous noradrenaline in the atrium. of dibenamine as well as the same effects of chlorpromazine marked
et al. (19)
noradrenaline.
heart
noradrenaline.
the
and Zaimis (18),
the
atrium than in the normal one.
et al. (16) that
from
the histological
from that
of the rate in response
of the guinea-pig's
the endogenous
mainly
in reserpinized
of the spontaneous
increase
in
from sixty to three hundred/
below fifty/min.
of the endogenous
The initial and transient
between
frequency derives
of stimulation
tion of the phase 3 _of the repolarization
potential
muscle of dog's heart, Hoffman
and not from that of the phase 3 of the repolarization, 2 is constant
in
effects may derive
as were shown by Withrington
Using the papillar
and the rate of stimulation
min, 2) the shortening
These
decrease
of the phase 1 and
shown by many authors (2-5), though
by reserpinization,
should also be considered. have
time of the action
The latter effect consisted of the prolongation
3, and not of the phase 2 of the repolarization. depletion
showed a considerable
and
that
ad
in the atria of 5 mg/kg
the conclusion
of the endogenous
that the norad
effects of the endogenous
the effects of the adrenolytics.
of adrenolytics
on the configuration
of the spontaneous
atrial
trans
membrane
potential
were studied comparatively
As has been shown in the previous lytics on the normal resulted
atrium
report,
in the untreated
was the prolongation
in the increase of the total duration,
depolarization reserpinized frequent
phase (1). atrium
appearance
of the notch.
of the depolarization
the application
a less prolongation However,
atria.
effect of the adreno
and the appearance
On the other hand,
produced
and reserpinized
the most pronounced
time which
of the notch in the
of the adrenolytics
of the depolarization
to the
time and a less
the increase of the total duration,
which
mainly derived from the prolongation of the phase 3 of repolarization, was observed. The prolongation of the phase 3 developed usually associated with the decrease of the rate, but it was also observed to 5 x 10-5 of dibenamine. rization
Since the reserpinization
phase of the atrial
adrenaline markedly
lessens the in the
even in the stage of initial transmembrane
prolongation
normal
atrium,
increase of the rate in response
of the animal
potential,
the depletion
of the depolarization
and augments
the
prolongs
time,
prolongation
the repola
of the atrial
which
nor
was observed
of the repolarization
by the adrenolytics. It may be concluded that the presence of the endogenous norad renaline in the atrium maintains the normal length of the repolarization time and op poses to the prolonging effect of the adrenolytics on the repolarization time. The reserpinized atrium in which spontaneous action potential had been abolished by the adrenolytics
was not restarted
was easily restarted
by the addition
atrium
almost
the repetitive rization
the same events have
by the washing-out of adrenaline
of the nutrient
or noradrenaline.
been observed.
However,
additions of either amine and washing-out depolarization
In the normal normal
atrium
recovered the prolonged
in the
repola
time to almost initial level, especially in the case of dibenamine,
of the prolonged
solution but
time, the depressed amplitude
but the recovery
and the decreased
resting
potential did not progress beyond the level in the stage II of the effects of the adreno lytics (1). On the other hand, the prolonged repolarization time of the reserpinized atrium caused by the adrenolytics recovery
was fully recovered
of the depolarization
ing potential
level associated
of the action potential
to the exogenous
atrial noradrenaline
the reserpinization
catecholamine.
and the sensitivity
No linear
of cat decreased correlation
with the
and the rest
to the level in the R-stage I of the effects of the adrenolytics.
and Zaimis (18) showed that heart
to the normal
time and amplitude
Withrington
the sensitivity
between
of the
the content
of the atria to exogenous noradrenaline
of
in guinea
pig was shown by Crout et al. (16). The hypersensitivity of the pressor response of the reserpinized cat to catecholamine was demonstrated by Burn et al. (20). The more ef fective reversibility of adrenaline or noradrenaline against the depressing effects of the adrenolytics on rate and configuration of the transmembrane potential in reserpinized atria coincides with the hypersensitivity of the reserpinized structure to either amine. The recovery of the depressed potential in the reserpinized atria by adrenaline or nor adrenaline was usually transient and the restarted action potential disappeared again at the variable time-length as well as in the untreated ones. The addition of l0' to 10-6of acetylcholine restarted the untreated (17) and the
reserpinized atria in which the spontaneous action potentials had been abolished by the adrenolytics. The addition of acetylcholine produced a full recovery of the resting potential, the amplitude and depolarization time of the action potential to the initial level. However, the prolonged repolarization time of the reserpinized atrium and the rate of the potential were not recovered to the normal level . Further, the recovering effect of acetylcholine was longer-lasting with the exception of the repolarization phase . The prolongation of the repolarization phase which had been effective to acetylcholine alone was restored effectively to the normal level by the combined addition of acetyl choline and noradrenaline. The addition of noradrenaline to the reserpinized atrium which had been recovered by acetylcholine as described above induced sometimes a sign of the hyperpolarization in the phase preceded with the repolarization one . It has been postulated that the heart responds to acetylcholine biphasically (7-14). The stimulating effect of acetylcholine has been believe to derive from the direct effect on the ganglion cell or chromaffin cell within the heart (7, 8) or from the release of adrenaline-like substance from the structure (9-11). The restarting and recovering effects of acetylcholine against the depressive action of the adrenolytics on rate and configuration of the atrial transmembrane potential in reserpinized rabbit show clearly the cholinergic mechanism of acetylcholine itself . The important role of the synthesis and liberation of the endogenous acetylcholine in the heart for initiation and maintenance of the spontaneous beat has been shown by Burn et al. (12, 13, 21, 22). They hypothe sized that the critical level of the endogenous acetylcholine in the heart was a deter mining factor for the initiation of the contractile stimulus and the addition of acetyl choline exhibited an inhibitory or excitatory effect according to the rate of synthesis of the endogenous acetylcholine. The restarting effect of acetylcholine on the reserpi nized atrium of which the action potential had been abolished by the adrenolytics might have been elucidated in association with the increase of the resting potential by acetyl choline or vagal stimulation, as reported by Burgen and Terroux (23), Hutter and Trautwein (24), and Hoffman and Suckling (25). From the results described above, it is assumed that the exogenous and probably endogenous acetylcholine regulates and maintains the depolarization phase of the atrial action potential, while adrenaline or noradrenaline does the repolarization phase . The less prolongation of the depolarization time produced by the adrenolytics in the reser pinized atrium than in the normal one is assumed to derive from the change of the rate of the content of the endogenous noradrenaline to acetylcholine, i.e. the overwhelming majority of the endogenous acetylcholine, by which the effect of the adrenolytics on the depolarization time is opposed. The results presented by Malhotra et al. (26) that the intravenous injection of 0.5 mg/kg of reserpine in dogs increased the extractable acetylcholine by 75.3% in the S-A node, 48.3% in the right atrium and 44.4% in the right ventricle support this assumption. In the unpublished experiment the author confirmed that the untreated rabbit's atrium in which the action potentials had been abolished by the application of a moderately high concentration of DO was not re
started by adrenaline or noradrenaline, but restarted by the addition of acetylcholine or the repetitive washing. However, the prolongation of the repolarization time pro duced by DCI was not recovered to the initial level even by these procedures. The results also support this assumption. The relation between the effects of adrenolytics and acetylcholine on the atrial potentials" will be discussed in details in the succeeding report. SUMMARY Effects of 5 x l0-' of dibenamine, 10-' of chlorpromazine and yohimbine were studied on the non-pacemaker potentials of the isolated atria of the rabbit pretreated with reserpine. Competitive effects of adrenaline, noradrenaline and acetylcholine against these adrenolytics were compared between reserpinized and untreated atria. 1. The action potentials in the isolated atrium of the reserpinized rabbit showed the considerable decrease in rate and the prolongation of the 90% repolarization time, especially the phase 3 of the repolarization. 2. The initial rate-increasing effect of dibenamine, and the rate-decreasing effect of dibenamine, chlorpromazine and yohimbine were manifested more markedly in the reserpinized atrium than in the untreated one. The lag time needed for the standstill of the atrium was shortened by the reserpinization. 3. The application of the adrenolytics produced less prolongation of the depola rization time and less frequent appearance of the notch on the depolarization phase in the reserpinized atrium than in the untreated one. More marked prolongation of re polarization phase, especially the phase 3, was observed in the reserpinized preparation. 4. The reserpinized atrium in which the spontaneous action potentials had been abolished by the adrenolytics failed to restarted by washing-out, but was easily restarted by adrenaline or noradrenaline as well as the untreated one. However, the repetitions of the addition either amine and washing-out recovered the depressed transmembrane potential more effectively in the reserpinized atrium than in the untreated one. Espe cially, the prolonged repolarization time of the reserpinized atrium was fully recovered to the normal level. However, the recovery by adrenaline or noradrenaline was also incomplete and transient in the reserpinized atria. The action potentials in the reser pinized atria disappeared again at the variable time-length after the addition as well as in the untreated one. 5. The addition of acetylcholine restarted the action potential which had been abolished by the adreno]ytics in the reserpinized atria. The decreased resting potential, the depressed amplitude and prolonged depolarization time of the action potential were completely recovered to initial level, but the prolonged repolarization time and decreased rate of the action potential failed to recover. The recovering effect of acetylcholine was longer lasting than that of adrenaline or noradrenaline. 6. The prolongation of the repolarization phase was effectively restored to the normal level by the combined addition of acetylcholine and noradrenaline. Occasionally,
the addition
of noradrenaline
produced
a hyperpolarization
at the phase following
the
repolar,ization. 7. regulates atrium,
From the results described and maintains while
above, it is assumed
the depolarization
noradrenaline
as a possibility
that acetylcholine
phase of the action potential
does the repolarization
in the rabbit's
one.
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