The Action of Acetylcholine and Epinephrine
on the Cellular Membrane Potentials and
Contractility of Rat Atrium
By J. LEYDEN W E B B , P H J D . AND PHILIP B. HOLLANDER
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Acetylcholine diminished the developed tension of contraction in rat atria; simultaneously there
was a slight increase in resting potential, no appreciable change in action potential, a decrease in
the rate of conduction, only a minor alteration of the form of contraction, and, most strikingly, a
decrease in the duration of the action potential or the area enclosed by it. The rate of repolarization
subsequent to the action potential was markedly accelerated. Epinephrine, in general, produced the
opposite effects and slowed the repolarization rate as it augmented atrial contraction. Cholinesterase
does not seem to play a direct role in cardiac membrane potentials or ion transfer since physostigmine had no effect of its own on the electric manifestations. Certain aspects of the mechanisms
involved are discussed.
T
minute in Krebs-Ringer-bicarbonate medium at pH
7.4 and 30 C. were determined simultaneously using
microelectrodes and a sensitive strain gage as
previously described.7
HE mechanisms by which acetylcholine
and epinephrine alter cardiac function
are perhaps related to changes in ionic
fluxes across the cell membranes and the resultant disturbances in electric potential. The
slight increase of the resting potential following
vagal stimulation 1 and the shortening of the
duration of the action potential 2 haA'e been
adequately confirmed by numerous workers
and recently placed on a more quantitative
basis by the use of intracellular electrodes,3-A
applying choline esters as well as vagal stimulation. Alterations in the action potential have
been variable but a decrease has most commonly been reported. The effects of epinephrine
on the electric characteristics of cardiac muscle
are less well understood and in general have
been assumed to be rather slight.6- • The purpose of the present work is to extend our knowledge in these directions in the attempt to
understand better the nature of the coupling
between the membrane action potential and the
contractile elements of the cardiac cell.
RESULTS
Effects of acelylcholine on tlic normal alrium.
Acetylcholine was applied in concentrations
between 10~7 M and 10~B M after an adequate
series of control readings had been made; a
second series of control readings was made after
the tissue had become stabilized following removal of the acetylcholine. The control readings given are means of these two since this
tends to eliminate any drift in the tissue characteristics during the procedure. The results are
given in table 1. The number of penetrations to
yield statistically valid results required 10 to
15 minutes following addition of acetylcholine;
the maximal action occurred between 1 and
V/l minutes and partial recovery ensued. The
overall action is given for the total interval and
for all the concentrations used. The maximal
action from the two or three penetrations made
during this period is also shown for all concentrations and for the higher range of concentrations. Although not as statistically valid as the
overall results, due to the fewer penetrations
possible, the results obtained for the maximal
action are very similar except more marked in
each category. It is also evident that the higher
acetylcholine concentrations produced greater
alterations in most cases.
METHOD
The membrane potentials and mechanical activity
of mt atria electrically stimulated at a rate of 200/
From the Department of Pharmacology, School of
Medicine, University of Southern California, Los
Angelas, Calif.
This work was supported by a grunt from the Life
Inmirunco Medical llesearch Fund.
Received for publication January 16, 105C.
332
Cimilalion Reteanh, Volume IV, Man I9S0
333
WEBB AND HOLLANDER
TABLE 1.—Effects
of Acetylcholine on Hat Atria.
Maximal Action (1 to 1H min.)
Overall Action (10 to 15 min.)
All Concentrations
Ach
Ach
Control
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Resting potential (mv.)
Action potential (niv.)
Overshoot (mv.)
Action potential—duration
(msec.)
Action potential—area (mv.
sec.)
Developed tension (mg.)
Developed
tension—duration (msec.)
Developed tension — rise
time (msec.)
Conduction time (msec.)
Conduction rate (em./soc.)
Excitation-con tract ion time
(msec.) '
Latent period (msec.)
Penetrations
Number of ruts
60.94
72.92
11.98
46.30
1.33
506.2
107.0
62.52
72.93
10.41
21.14
0.644
366.2
103.6
% Change
Control
+2.6
-54.4
59.59
72.16
12.57
48.01
-51.5
1.32
0.0
-27.6
-3.2
547.0
114.5
62.38
73.35
10.97
17.05
0.532
325.5
10S.2
Ach - 6-10 X 10"
% Change
+4.7
+ 1.7
Control
-64.5
57.91
69.27
11.36
50.36
-60.0
1.35
-40.6
-5.5
566.8
115.0
Ach
62.26
71.79
9.53
11.32
0.365
297.1
109.6
i\l
% ChanRc
+ 7.5
+3.6
-77.5
-72.9
-47.6
-4.7
44.94
42.95
-4.4
47.63
43.21
-9.3
46.51
41.87
-9.9
14.76
73.2
10.35
14.91
72.4
9.89
+ 1.0
15.44
69.9
9.90
15.78
68.5
9.S3
+2.2
14.88
72.6
9.S9
15.79
68.4
10.17
+6J
-1.0
-4.4
24.SO
-1.2
25.61
+ 1.1
25.96
+4.S
25.H
429
20
293
There was a slight, though significant, increase in the resting potential, this occurring in
17 out of the 20 preparations, demonstrating
the Caskell effect in rat atrium.1 There was no
certain effect on the action potential unless the
slight increases noted during maximal action
are significant; at least the absence of a depression seems certain. The absolute level of
depolarization reached was thus somewhat less
in the presence of acetylcholine, as measured by
the decrease in the overshoot, but it is doubtful
if this is of any importance in altering the
mechanical response. The most striking change
observed was the decrease in the duration of the
action potential and the area enclosed by it.
This was almost entirely due to the more rapid
rate of repolarization induced by acetylcholine;
the rise time of the action potential was only
about ]/100th the total duration so that any
shortening of it could not have had appreciable
effect. The acceleration of the repolarization
rate was approximately twofold when the contractile depression was 25 per cent and fivefold
when the contractile depression was 50 per
cent; higher concentrations of acetylcholine
produced up to tenfold accelerations of repolarization accompanied by marked reduction of
contraction.
25.34
229
20
43
-2.2
-0.7
24.77
121
9
-6.1
+2.S
2S
The rise time and duration of the contraction
were shortened relatively little, indicating that
the form of the total contraction was not altered
appreciably by acetylcholine although a slight
shift of the peak may have occurred. Some
shortening would be expected during the depression of developed tension and it would
appear that in the rat atrium there is no specific
failure of slowly reacting cells as postulated by
Burgen and Terroux1 in the cat atrium, but
only a decreased contraction of each cell.
The effect of acetylcholine on conduction
rate was very slight but is believed to be significant since it occurred in 15 out of the 20
preparations and was more marked with the
higher concentrations of acetylcholine. It is
surprising that the marked shortening of the
action potential had so little effect on the conduction rate and indicates that the factors
involved in conduction probably depend primarily on the magnitude of the depolarization
and the responsiveness of the normal membrane to depolarization. The slight changes
observed in the latent period are probably not
significant since half the preparations showed
and increase and half a decrease.
The characteristic effects of acetylcholine
seem to be independent of the substrate from
334
ACETYLCHOLINE AND EPINEPHRINE ACTIONS ON RAT ATRIUM
which the cardiac muscle is deriving its energy.
Atria allowed to beat in the absence of substrate show a steady decline in developed tension to low values; if either glucose or pyruvate
is added at this time, recovery is observed. In
such atria beating in the presence of glucose or
pyruvate, and presumably deriving most of
their energy from these substrates, the effects
of acetylcholine were quite comparable to those
observed on fresh atria utilizing endogenous
substrate.
Effect of •physosligmine on the response to ace-
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tylcholine. In the presence of 10"1 M physostigmine, the sensitivity to acetylcholine was
increased over 100 fold. The results of a single
experiment are shown in table 2 and, although
not quantitatively reliable, they demonstrate
the same changes noted with acetylcholine
alone, except to a greater degree. The depression of the magnitude of the action potential
began when the action potential had been
shortened markedly, as if the repolarization
process were beginning before the membrane
had time to depolarize normally, and similar
depressions of the action potential were observed with very high concentrations of acetylcholine and carbachol when the repolarization
rate had been greatly accelerated. More asymmetry of the contraction may be noted, the
TABI,E 2.—Effects
of Acetylcholine in the Presence of Physostigmine, of Physostigmine and
of Carbachol.
Ach (10-« M) after
physostigmine (10"< M)
Control
Resting potential (mv.)
Action potential (mv.)
Overshoot (mv.)
Action potential—duration
(msec.)
Action potential — area
(mv. sec.)
Developed tonsion (mg.)
Developed tension—duration (msec.)
Developed tension — rise
time (msec.)
Conduction time (msec.)
Excitation-contraction
time (msec.)
Latent period (msec.)
Penetrations
Number of rats
peak occurring relatively earlier than in the
control, and there was definite, although slight,
slowing of the rate of conduction.
Effect of physostigmine alone. Physostigmine
produced a slow depression of rabbit atria, 4 due
presumably to accumulation of acetylcholine
produced endogenously, and recently such results have been used as evidence for the role of
acetylcholine in cardiac contraction. 9 ' 10 The
possible role assumed by some investigators for
cholinesterase in ion transport across membranes also prompted us to determine if physostigmine exerted any direct effect on the electric
behavior of the rat atrium. No immediate
effects were observed from 10~6 M to lO"1 M
physostigmine. After several minutes a slow
depression of the developed tension was seen,
reaching a steady level, and the changes in the
atria at this time are given in table 2. The
results are compatible with a mild acetylcholine effect. It is further interesting that these
effects were produced by 10~6 M physostigmine and increasing the concentration to 10~'
M resulted in no additional action.
Effect of carbachol. Carbachol differs primarily from acetylcholine in being resistant to
cholinesterase and hence is somewhat more consistent and stable in its cardiac action.8 The
results from seven experiments are given in
65.55
76.11
10.56
44.55
1.21
3S2.3
117.3
Ach
65.70
68.50
2.SO
8.25
0.269
190.2
107.5
% Change
+0.2
-10.0
-81.4
-77.8
-47.9
-8.4
PhysoJtigmine (10"*-l 0"» M)
Carbuchol (10 ' - 1 0 ' M)
Control
Physo
% Change
Control
Cch
5S.S6
69.92
11.06
51.26
59.98
71.92
11.94
43.55
+ 1.9
+2.9
-15.0
59.73
70.04
10.31
48.90
60.74
69.54
8.SO
21.27
-13.3
1.29
1.505
504.7
115.5
1.305
429.1
117.9
-15.0
+2.1
472.8
102.4
0.614
% Change
+ 1.7
-0.7
-56.5
-52.5
303.1
94.63
-35.9
-7.6
50.11
40.25
-19.7
50.05
48.55
-3.0
49.61
44.33
-10.6
14.55
7.77
15.25
5.75
+4.8
14.58
11.26
13.95
9.S5
-4.3
-12.5
12.S7
-26.0
12.46
10.42
-3.2
-3.0
22.32
21.00
-5.9
25.84
23.SO
-7.S
17
23.61
109
7
22. SS
SI
-3.1
23
2
18
1
S
10.74
335
WEBB AND HOLLANDER
TABLE 3.—Effects
of Epiiiephnne and the ftemoval of Acetylcholiiie.
Epinephrine (5 X 10"' M)
Control
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Itcsting potential (rav.)
Action potential (mv.)
Overshoot (mv.)
Action potential — duration
(msec.)
Action potential—urea (mv.
sec.)
Developed tension (mg.)
Developed tension — duration
(msec.)
Developed tension — rise time
(msec.)
Conduction time (msec.)
Excitation - contraction time
(msec.)
Latent period (msec.)
Penetrations
Number of rats
Epi
% Change
Control
Ach
-4.5
-3.8
62.61
76.08
13.47
42.17
63.39
75.12
11.73
19.07
60.37
68.65
8.28
41.22
57.66
66.07
8.41
46.57
+ 13.0
1.12
1.34
+ 19.6
445.2
105.8
576.9
108.9
Acetylcholine removal (10"*-10~«
+29.5
+2.9
1.085
424.2
109.4
Post-waih
0.516
305.2
104.1
% ChanRC*
62.24
74.86
12.62
49.96
+18.5
1.27
+ 17.0
544.3
113.5
-0.6
-1.6
+28.3
+3.7
46.84
50.65
+8.1
46.30
43.68
49.96
+7.9
13.30
10.42
13.64
9.53
+2.6
-8.5
16.08
10.90
16.72
10.17
17.27
8.98
-17.6
23.72
23.17
-2.4
26.25
-2.7
76
50
7
26.98
142
8
26.89
132
+7.4
37
* Calculated from the control levels.
table 2, from which it may be seen that the
changes produced were quite similar to those
produced by acetylcholiiie.
Response to removal of acetylciwline. When
acetylcholine is washed out, following an exposure of 10 to 15 minutes, there ensues a
temporary stimulation of atrial contractility
above the control levels* and it was of interest
to determine the changes in electric behavior
during this period. It was difficult to obtain
more than four to five penetrations during the
two to three minutes of heightened contractility, but the averages from eight preparations
showed some definite changes (table 3). The
rise in developed tension was accompanied by a
prolongation of the action potential, or a slowing in the rate of repolarization, without significant changes in the resting or action potentials. A slowing of the conduction rate occurred
in seven out of the eight preparations. This
increase in conduction time accounted for the
decrease in the excitation-contraction time7 so
that the latent period was essentially unchanged .
Effect oj epi?iephrine. The action of epinephrine was in general the opposite to that of
acetylcholine on the characteristics measured
(table 3). The rise in developed tension was
accompanied by a slowing of the repolarization
rate and slight depression of both the resting
and action potentials. The rate of conduction,
however, was slightly slowed, as it was with
acetylcholine, substantiating the independence
of conduction and the duration of the action
potential. The slowing most likely resulted
from the lowered membrane potentials.
DISCUSSION
The shortening of the action potential duration produced by vagal stimulation or choline
esters has been reported several times, using
both contact electrodes and intracellular electrodes, but in no case has the correlation
between the contractile depression and this
shortening been expressed quantitatively, and
hence it is impossible to compare the present
results with those obtained in other species. The
only demonstrable marked effect of either acetylcholine or epinephrine on the membrane
electric properties of the cardiac cells was upon
the rate of repolarization subsequent to the
development of the action potential and the
obvious correlations tempt one to assume a
causal relationship. However, it must be realized that the shortened action potential may
not be the cause of the diminished contraction,
in the case of acetylcholine, but may be the
result of an independent action, or an unimportant coincident result of the same action, or
the result of the diminished contraction itself.
336
ACETYLCUOLINE AND EPINEPHRINE ACTIONS ON RAT ATRIUM
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The data of the present study cannot be used
to decide between these but results to be published from modifications of atrial activity by
other means strongly argue against the last
two possibilities.
The alterations in the resting and action membrane potentials by acetylcholine and epinephrine, slight though they are, and probably
without significance in the contractile effect,
are believed to be of great importance in the
eventual explanation of the mechanisms involved. The concept that acetylcholine causes a
large nonselective increase in ion permeability,
such as has been postulated to explain the endplate depolarization in skeletal muscle,11 certainly cannot be valid for cardiac muscle.
Rather there would appear to be a selective
increase in permeability to a particular ion
following depolarization of the membrane,
whereas the permeability properties of the normal polarized membrane do not seem to be
greatly affected. The possibility is suggested
that there are fixed charges of various types in
the membrane and that some of these at least
are intimately dependent on metabolic processes. Furthermore, these groups control in
part the diffusion rates of ions through the
membrane. Reaction of acetylcholine or epinephrine specifically with certain of these
charged groups may modify the permeability
in the manner suggested by the present results.
Discussion of this hypothesis must await further studies on the modifications of cardiac
function by a variety of means.
SUMMARY
The actions of acetylcholine, carbachol, physostigmine and epinephrine on atrial contraction and membrane potentials were investigated. The choline esters increased the
membrane potentials, but only slightly, while
markedly shortening the duration of the action
potential by an acceleration of the rate of
rcpolarization. Epinephrine produced opposite
actions. The possibility of correlating these
changes with the altered contractility was discussed. No evidence for a direct role of cholinesterase in ion transport across the cardiac
membrane was found.
SUMMARIO IN1 INTERLINGUA
Le action de acetylcholina, carbachol, physostigmina, e epinephrina super le atrial contraction e potentiates membranal esseva investigate. Le esteres cholinic augmentava le
potentiales membranal, sed solmente a leve
grados, durante que illos reduceva marcatemente le durantia del potential de action per
accelerar le repolarisation. Epinephrina produceva le effecto contrari. Es discutite le
possibilitate de correlationar iste alterationes
con le alterate contractilitate. Esseva trovate
nulle indicio de un rolo directe de cholinesterase
in le transporto de iones a transverso le membrana cardiac.
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The Action of Acetylcholine and Epinephrine on the Cellular Membrane Potentials and
Contractility of Rat Atrium
J. LEYDEN WEBB and PHILIP B. HOLLANDER
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Circ Res. 1956;4:332-336
doi: 10.1161/01.RES.4.3.332
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