Relation between Intracellular Sodium and Twitch Tension in Sheep Cardiac Purkinje Strands Exposed to Cardiac Glycosides
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697
Relation between Intracellular Sodium and Twitch
Tension in Sheep Cardiac Purkinje Strands Exposed to
Cardiac Glycosides
J. A. Wasserstrom, D. J. Schwartz, and H. A. Fozzard
From the Cardiac Electrophysiological Laboratories, Departments of the Pharmacological and Physiological Sciences and of
Medicine, The University of Chicago, Chicago, Illinois
SUMMARY. Changes in intracellular sodium ion activity (ajja) produced by several cardiac glyco-
sides were correlated with twitch tension in sheep cardiac Purkinje strands. Simultaneous measure-
ments of aW. and twitch tension were obtained through the use of Na-sensitive intracellular
microelectrodes (ETH 227) in Purkinje preparations stimulated at a frequency of 1 Hz. All concen-
trations of ouabain, acetylstrophanthidin, and actodigin that were tested caused an increase in aWa
immediately before, or coincident with, a positive inotropic effect. No fall in aWa was observed at any
positive inotropic concentration of digitalis in these beating fibers. In all cases, the onset and washout
of the positive inotropic effect were paralleled by the rise and fall inaNa, respectively. No dissociation
between changes in aka and twitch tension occurred at any concentration of any of the agents used.
The relation between changes in a'Na and twitch tension was linear with 1 HIM increase in aWa
producing about a 100% increase in the twitch magnitude. Propranolol did not significantly alter this
relationship. The increase in aiMa with digitalis was also associated with a reduction in the maximum
depolarization rate of the action potential, presumably as a consequence of a reduction in the
transmembrane Na electrochemical gradient. These results indicate that the positive inotropic action
of digitalis in sheep Purkinje strands is always associated with a rise in aNa secondary to inhibition
of the Na pump. This increase in aNa could increase calcium available for contraction via the Na-Ca
equilibrium exchange process. In addition, the increase in aka reduces VmM, as a consequence of
decreasing the electrochemical gradient for Na. (Circ Res 52: 697-705, 1983)
THE mechanism of action of digitalis has been the pounds. This suggests also that pump inhibition and
topic of much research in recent years. Biochemical positive inotropy are dissociable. Several investigators
and physiological evidence has indicated that the have reported a stimulation of Na and K countertrans-
enzyme responsible for Na and K countertransport port with therapeutic concentrations of glycosides
across the sarcolemma (Na,K-adenosine triphospha- (Cohen et al., 1976; Godfraind and Ghysel-Burton,
tase or Na,K-ATPase) serves as the receptor for car- 1977; Deitmer and Ellis, 1978, Ghysel-Burton and
diac glycosides (for review, see Schwartz et al., 1975). Godfraind, 1979). These conflicting results may be
It has been suggested that digitalis binding to this the consequence of methodological and species dif-
receptor interferes with its normal functioning, thus ferences (Grupp et al., 1982), but, as yet, there has
allowing Na+ to accumulate in the sarcoplasm, with been no comprehensive explanation for the apparent
a concomitant loss in cellular K+. This increase in discrepancies in these recent reports. There is some
cellular Na+ might then interfere with the normal biochemical support for the proposal of digitalis stim-
Ca++ efflux process via Na-Ca equilibrium exchange ulation of the Na,K-ATPase. However, other possible
(Reuter and Seitz, 1968). In this manner the accumu- explanations exist; for example, digitalis may cause
lation of Na+ in the sarcoplasm could cause a rise in release of catecholamine in isolated tissues (Seifen,
the amount of calcium available for contraction (Lan- 1974). The catecholamines in turn, stimulate the Na
ger and Serena, 1970). This scheme is popularly in- pump and cause the observed fall inaka (Wasserstrom
voked to relate the apparently selective binding of et al., 1982).
digitalis to the Na,K-ATPase to its powerful positive One of the difficulties in interpreting the above
inotropic action. results is that Na pump function has been inferred
Recently, however, evidence has been presented from the rate of rubidium-86 transport as a substitute
that appears to dissociate Na pump inhibition from for external K+, or from the Na+ and/or K+ content
the therapeutic action of cardiac steroids. Okita and as determined chemically. None of these methods is
co-workers (1973a, 1973b) found biochemical and entirely suited to a direct correlation between changes
electrophysiological indications of a dissociation be- in intracellular ionic composition and the positive
tween these actions of digitalis. Mendez et al. (1974) inotropic action of digitalis in intact cardiac tissue.
reported that a semisynthetic glycoside, actodigin, has The use of Na-sensitive intracellular microelectrodes
a greater margin of safety than conventional com- (Na-ISE) has recently made these measurements pos-
Downloaded from http://circres.ahajournals.org/ by guest on March 6, 2015698 Circulation Research/Voi. 52, No. 6, June 1983
sible. Lee et al. (1980) demonstrated that intracellular potential was recorded with a microelectrode filled with 3
Na ion activity (a^a) increased at the time that dihy- M KCI (tip resistance of 5-15 mS2). The Na-ISE were made
dro-ouabain had produced a positive inotropic action from the same blank micropipettes (WP instruments, bo-
in dog Purkinje strands. The strength of contraction rosilicate capillary tubing), but were siliconized so that their
was measured periodically throughout exposure to tips would hold the Na-sensitive neutral ligand ion exchan-
and washout of the glycoside. Lee and Dagostino ger resin ETH 227 (courtesy of Drs. Simon and Ammann,
Zurich). The silanization process has been described else-
(1982) used Na-ISE in constantly stimulated canine where (Sheu and Fozzard, 1982). Only Na-sensitive elec-
Purkinje fibers and observed a close relation between trodes with slopes greater than 48 mV per 10-fold increase
changes in aWa and tension during onset and washout in Na+ were used. Each microelectrode was calibrated be-
of several concentrations of strophanthidin. However, fore and after every experiment, using mixed solutions of
Deitmer and Ellis (1978) showed that low concentra- NaCl and KCI with a constant total ionic strength of 300
tions of strophanthidin that, presumably, are posi- mM. Transmembrane potential was measured with a Pico-
tively inotropic cause a fall in aisia in quiescent sheep metric Instruments electrometer, and the Na-ISE potential
Purkinje fibers. Lee and Dagostino (1982) also found was measured with a Keithley model 604C high impedance
some indications of Na pump stimulation but did not electrometer. The maximum depolarization rate (Vmai) was
fully examine this phenomenon. The present study measured by an electronic differentiator. Potential and
twitch tension measurements were displayed on a Tektronix
was designed to examine the effects of low and high 5113 storage oscilloscope and Gould Brush 220 chart re-
concentrations of several cardiac steroids in regularly corders for data recording. Microelectrode measurements
stimulated cardiac fibers to determine the exact direc- were also displayed on digital panel meters, and these
tion and magnitude of changes in a'^a and to relate values were used for calculation of a^a-
these changes to the inotropic actions of several Once stable impalements were obtained, as indicated by
agents. three identical measurements of a^a, one of three cardiac
The other aspect of this study was to examine some steroids was added to the superfusate. Ouabain, acetylstro-
of the physiological consequences of changes in the phanthidin (AS), and actodigin were prepared in concen-
intracellular ionic composition. Eisner et al. (1981) trated stock solutions of 5% ethanol. Each agent was diluted
examined the relation between aNa and twitch tension in Tyrode's solution to the proper concentration immedi-
ately before use in each experiment. The concentrations
in sheep Purkinje fibers under voltage clamp. Na used were as follows: ouabain, 1-4 X KT7 M; AS 0.2-1.3 X
pump inhibition was accomplished by exposure to 0 10~7 M and actodigin 0.6-1.9 X 10"6 M. LOW and high
K+ solutions. Their conclusion was that the positive concentrations of each agent were tested so that the time
inotropic action of 0 K+ solutions was the result of course of changes in a^a and twitch tension could be ex-
Na+ accumulation and Na-Ca exchange, thus provid- amined.
ing a good basis for comparison with digitalis inhi- The Na-sensitive resin ETH 227 has a selectivity of about
bition of the pump. Brown et al. (1981) have related 50:1 for Na:K. Because of the high electrode impedance, the
the transmembrane gradient of Na+ to the magnitude noise on these signals is greater than on KCl-filled pipettes,
of the fast inward Na+ current in internally perfused making the digital voltmeter reading a useful one for mon-
isolated ventricular myocytes. Our measurements of itoring the stability and for determining the voltage differ-
aka and the Na+ gradient across the sarcolemma allow ence for comparison with calibration curves. Because drift
of 1-2 mV can occur over the 6-8 hours of these experi-
us to relate the maximum depolarization rate of the ments, we only used data from experiments where contin-
action potential (Vma*) to changes in the Na+ gradient uous recordings were available, usually with after-control
as a result of Na+ pump inhibition. periods. Individual measurements are easily made to ±0.5
mV, which corresponds to about ±0.2 mM aNa in the range
Methods of values obtained.
Our method for measuring aiMa in stimulated preparations Continuous stimulation at a constant frequency is desir-
has been discussed in detail elsewhere (Cohen et al., 1982; able during these experiments in order to obtain reliable
Wasserstrom et al., 1982). Briefly, Purkinje strands were and reproducible measurements of twitch tension. In addi-
dissected from sheep hearts obtained fresh from the local tion, the positive inotropic effect of digitalis is frequency-
slaughterhouse. One end of the fiber was pinned to the dependent. Because the electrical response time of these
floor of the beeswax-paraffin tissue chamber through which Na-ISE is slow (about 500 msec), it was not possible to
Tyrode's solution was flowing (10 ml/min, 37°Q. The measure aWa accurately during the cardiac action potential.
composition of the solution was (in mM): NaCl, 137; KCI, Our measurements were obtained during a brief pause (5-
5.4; NaHCO3, 22; MgCl2, 1; NaH2PO4 2.4; CaCl2, 2.4; and 15 seconds) every 5 minutes during the control, drug, and
dextrose, 5.5. The solution was gassed constantly with a washout periods. This procedure allows accurate measure-
mixture of 95% O2, 5% CO2 to maintain oxygenation and ments of aka during a brief diastolic interval.
pH (7.35). One end of the fiber was pinned to the floor of Sample means were compared using Student's t- test, and
the test chamber; the other end was attached to a force the level of signficance was considered to be P < 0.05.
transducer (based on a Texas Instruments model 138 infra-
red photodiode) by a hooked stainless steel needle. Extra- Results
cellular stimulation was delivered through silver wires in-
sulated except at their tips at a frequency of 1 Hz (0.5 msec, Relation between a^a and Tension
1.5 X threshold). The force transducer was mounted on a
micromanipulator so that the fiber could be stretched to The time course of the changes in al\ia and twitch
about 50% of L,,^ (Wasserstrom et al., 1982). tension may be important in examining the relation
After about 1 hour of stimulation, the fiber was impaled between these two actions of digitalis. Figure 1 shows
with a conventional and a Na-ISE. The transmembrane the effects of ouabain on the force of contraction and
Downloaded from http://circres.ahajournals.org/ by guest on March 6, 2015Wasserstrom et al./ Relation between Naj and Twitch: Digitalis Effects 699
A 30—
B
Ouabain 18
4xlO 7 M
4 s
._ 2
D
25 T
20
o 1.0-
c
o
b
0.1
10 20 30 40 50 60 10 20 30 40 50 60
Time (minutes) Time (minutes)
FIGURE 1. Panel A: effects of ouabain on afia and twitch tension in a beating sheep Purkinje strand. Stimulation at 1 Hz was maintained
except for brief interruptions to measure a NO- Prior to time 0, three identical values of afia and twitch tension were obtained. Ouabain (4 X
10~7 M) was added to the superfusate at time 0. Filled circles indicate twitch tension and triangles indicatea&a, in this and all subsequent
figures. Panel B: semilogarithmic plot of the same data presented in panel A. The linear portion of this curve occurred after 15 minutes of
exposure to ouabain.
a^a in a sheep Purkinje strand. After three identical tween changes in aha and the twitch. Acetylstrophan-
measurements of aha were obtained (not shown), 4 thidin is an ideal agent for this purpose, and it also
X 10~7 M ouabain was added to the superfusate. No has properties similar to strophanthidin, which has
effect on Na or twitch occurred within the first 10 been reported to cause Na pump stimulation in low
minutes. At the end of 10 minutes, aha began to positive inotropic concentrations (Deitmer and Ellis,
increase gradually. Five minutes later, twitch tension 1978). Figure 2A shows the effects of 1.1 X 10"7 M AS
began to rise. Thereafter, both aha and twitch in- on aha and twitch strength in a sheep Purkinje prep-
creased in a parallel fashion. Note that during the aration. This fairly high concentration of the aglycone
initial exposure there was no decrease in aha which caused an increase in aha and twitch within 5 minutes
would indicate a stimulation of Na pump activity. At after the addition of the AS to the superfusate. The
the time of positive inotropy, there had already been rapid onset of the inotropic action was paralled by the
an increase in aha- In this experiment, the increase in rise in aha- Both aha and twitch reached their maxi-
aNa occurred immediately before onset of the positive mum values at about the same time, about 35-40
inotropic action of the ouabain. Panel B of this figure minutes. Drug-free solution then was introduced, and
shows a semi-log plot of these two curves. The slopes the washout of the drug effects was examined. Within
of the change in aha and tension with time are identical 5 minutes, aha began to decrease, followed immedi-
for the linear portions of these two curves, indicating ately by a fall in twitch tension. Both aha and twitch
that their time constants were also identical (20 min- fell with nearly the same time course back to their
utes for aka/ 20.1 minutes for twitch). This parallel initial values within 30-35 minutes, with aha returning
between the rise in aha and positive inotropy was to control slightly faster than the twitch. This exper-
observed in all 11 fibers exposed to 1-4 X 10~7 M iment demonstrates that AS produces parallel changes
ouabain. in Naj and twitch tension during the onset of the
Ouabain thus caused a rise in aha and contractility positive inotropic action of digitalis similar to that
with the same time course, but with a very slight lag seen with ouabain. In addition, the washout of these
of twitch tension behind the increase in aNa. The effects of AS also shows a parallel fall in aha, with the
effects of this agent do not wash out readily in this change in aha often slightly leading the fall in twitch
preparation. Therefore, we tested the effects of other tension. These results were obtained in all 19 fibers
cardiac steroids whose rapid and reversible actions exposed to moderate concentrations of AS (0.9-1.3
allow more complete examination of the relation be- X 10~7 M).
Downloaded from http://circres.ahajournals.org/ by guest on March 6, 2015700 Circulation Research/Voi. 52, No. 6, June 1983
• ASUxl6 7 M -AS
i
A A A A A 18 _
16 |
A A
A A A A A 12 ~b
10
12
10
„ 8
60 90
Time (minutes)
B
• AS Ux 10 M
A Washout
I8
t
10 II E 3 M B 16 17 B
FIGURE 2. Panel A: effects of acetylstrophanthidin (AS) on a'Na and twitch tension in a Purkinje strand. After three identical readings for
aha and tension, AS (1.1 X 10 ~7 M) was added to the superfusate (time 0, first arrow). Washout with drug-free Tyrode's solution was begun
after 45 minutes of exposure to AS (second arrow). Panel B: graph of the relation between afia and twitch tension for the experiment as in
panel A. Filled circles indicate this relation during the onset of the positive inotropic effect of AS. Unfilled triangles represent this relation
during drug washout.
The relation between tension and aka for this ex- has a very rapid action and its effects are easily
periment is plotted in Figure 2B. The onset curve washed out. If its positive inotropic actions are to
(solid circles) describes a linear relation between a^a some extent independent of Na pump inhbition, as
and twitch tension. During washout (triangles), the has been suggested by some investigators (Mendez et
fall of tension and a^a is also linearly related. How- al., 1974), then we would expect some increase in
ever, there is some displacement to the left of the twitch force before any change in aha occurred. Figure
washout curve, indicating a slightly different relation 3 shows the effects of 1.9 X 1(T6 M actodigin on a
between the twitch and alvia during the two phases of sheep Purkinje fiber. a'Na and twitch tension remained
drug action. This hysteresis has also been observed constant until nearly 10 minutes after addition of the
under conditions of inhibition of the Na pump by 0 drug to the superfusate. After 10 minutes, both twitch
K+ solutions (Eisner et al., 1981). tension and aka began to increase and thereafter both
The semi-synthetic cardiac glycoside actodigin also increased rapidly until drug-free solution was super-
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+ Actodigin •AS •AS
-Actodigin -AS
l.9xlO6M 20 2.2xl6 8 M
1 i
15
i 12
II E
-
io - o
40
30
I 20
° ,0 20 40 60 80 100 120 140
Time (minutes)
FIGURE 4. Effects of threshold concentrations of AS on aha and
10 20 30 40 50 60 70 tension. At time 0, a low concentration of AS (2.2 X 10 " 8 M) was
added to the superfusate without causing a positive inotropic effect.
Time (minutes) After 60 minutes, the concentration of AS was doubled (4.4 X 10 ~8
FIGURE 3. Effects of the semi-synthetic cardiac glycoside actodigin M), which was just sufficient to produce a suprathreshold effect on
on aAaand twitch tension. Actodigin (1.9 X 1 0 ~ 6 M ) was added to twitch tension. Drug-free Tyrode's solution was superfused after
the superfusate at time 0, and washout was begun after 40 minutes the positive inotropic action was fully expressed.
of exposure to drug (arrows).
both the presence and the absence of propranolol
fused after 40 minutes. During washout of these (10" 6 M).
effects, aka and twitch tension decreased rapidly and Low concentrations of actodigin were also tested
reached their initial values after 20-25 minutes of for their effects on the relation between contractile
washout. Neither phase of this experiment demon- force and aka- Figure 5 shows the effects of 7.6 X 10~~7
strated any dissociation between the changes in aka M actodigin. aka and twitch tension both increased
and twitch tension. These results were obtained in all after 10 minutes of exposure to this concentration.
nine preparations exposed to 1.0-1.9 X 10~6 M acto- There was no indication of a fall in aka initially, either
digin. before or during the onset of the positive inotropic
We have thus far dealt only with the effects of high action of this concentration of actodigin (Cohen et al.,
concentrations of these three cardiac steroids. All 1976). During washout, there was no separation be-
three agents demonstrated the same relationship be- tween the recovery of aka and that of twitch tension.
tween aka and twitch tension during both onset and These actions were tested in 12 fibers with concentra-
washout of their positive inotropic effects. However, tions of actodigin in the range of 6-8 X 10~7 M, either
Na pump stimulation and dissociation of pump inhi-
bition from positive inotropy may be demonstrable
only at low positive inotropic concentrations of digi- Actodigin
-Actodigin
talis. Figure 4 shows the effects of a threshold positive
inotropic concentration of AS on twitch and Nai.
Propranolol was present in the superfusate starting
sixty minutes before AS was added. At time 0, 2.2 X
10 M AS was added to the superfusate. There was
no measurable change in twitch tension throughout
the subsequent 60 minutes of exposure to this agent.
No change in aka occurred in either direction during
this period. When the concentration was doubled to
4.4 X 10~8 M AS, a doubling of tension occurred that 3 4
c
coincided with an increase of about 2 mMaka- Both o
«s
a'Na and tension returned to their initial values after
removal of the AS from the superfusate. This exper- I
iment demonstrates that low concentrations of AS
that do not increase twitch tension have no effect on 20 30 40 50 60 70 80
Nai. Furthermore, these and slightly higher concentra- Time (minutes)
tions that do exert a positive inotropic action were FIGURE 5. Effect of a low concentration of actodigon on afja and
never observed to cause a decrease in Naj. These twitch tension. At time 0, actodigin (7.6 X 10 " 7 M) was added to the
results were obtained in all 17 preparations treated superfusate. Washout was begun after 45 minutes of exposure to
with low concentrations of AS (2.2-6.6 X 10~8 M) in the drug.
Downloaded from http://circres.ahajournals.org/ by guest on March 6, 2015702 Circulation Research/VoJ. 52, No. 6, June 1983
in the presence (four experiments) or absence (eight 600 r
experiments) of 10~6 M propranolol.
The characteristic changes of aka and tension and
the overall relation between twitch tension and Nai 500
are summarized in Table 1. All results for various
concentrations of the three cardiac steroids have been A Washout
combined in this table because there was no differ- p 400 y=l22.3X-35.6
ence between the relation of tension to aka for these r=0.99
agents (as determined by an analysis of variance).
Experiments in which propranolol was added are not o 300
to
included in this table. The relation is more apparent c
in Figure 6 where these changes in twitch tension are A •
plotted as a function of changes in aka- Standard error
bars have been omitted for clarity of the illustration. *~ 200 • + Cardiac Glycoside
In this figure, the curve showing the onset of action A •
y= 117.5 X-38.3
is denoted by the filled circles. The hollow triangles r=0.99
show this relation during washout. This relation is 100
linear during both phases of drug action. The slope
of the onset line is 1.17, which indicates that a 1 mM
increase in ak a is associated with approximately a
doubling of the initial tension. The washout line has
nearly the same slope (1.22), which indicates that the
relation between twitch and aka is nearly the same for FIGURE 6. Relation between a ^ and twitch tension for alJ three
the two phases of drug action. There is a very slight cardiac glycosides. This figure summarizes the data in Table 1.
shift of the washout curve to the left of the onset Only mean values are included for the clarity of the illustration.
curve which is not significantly different. This may Equations refer to the regression lines for onset (filled circles) and
reflect a hysteresis that was occasionally observed washout (triangles), respectively.
between the two phases in some experiments (Fig.
2B). However, the overall relation seems to be nearly fibers used in this series of experiments was 7.6 ±
superimposable for the two phases of the experi- 0.62 mM (n = 31). The doubling ofaka that occurs at
ments. these concentrations of cardiac glycosides would
markedly reduce the inward driving force for Na + .
Relation between aka and Maximal Upstroke Consequently, Vmax would be expected to decrease
Velocity with the fall in the Na + electrochemical gradient
The changes in aka induced by positive inotropic (A/iNa) caused by the increase inak a . Figure 7 shows
concentrations of digitalis might be expected to have the effects of changes in AjiNa on Vmax. Results were
additional influences on the normal electrophysiolog- obtained only from those fibers that depolarized 2
ical functioning of the heart. One possible conse- mV or less during the course of the experiment.
quence of increasing aka would be to decrease the AjtiNa was calculated assuming that V m was —16 mV,
electrochemical gradient for Na + across the sarco- which has been experimentally derived as the voltage
lemma. The normal aka for the sheep cardiac Purkinje at which Vmax is greatest in sheep Purkinje fibers
(Walton and Fozzard, 1983). It is assumed that this
TABLE 1 value did not change with the concentrations of car-
Relation between Aa'Na and A% Twitch Tension (A%T) for All diac steroid used in these experiments and with a cell
Three Cardiac Steroids
depolarization of 2 mV or less. The initial value of
Onset Washout Vmax for these experiments was 527 ± 15.9 V/sec (n
= 17). The slope of the relation between V max and
Aak,, Aakfa A/iNa is indicated by the solid line. This relation
n (mM) AS6T (miu) A' n closely approximates the theoretical curve (dotted
31 0 ±0 0± 0 4.50 ± 0.82 512.1 ± 7.9 5 line) expected if (1) Vmax is a direct indicator of Na +
29 0.85 ±0.16 49.9 ± 2.3 3.55 ± 0.15 395.5 ± 4.5 2 inward current (INB; Walton and Fozzard, 1979) and
31 1.35 ±0.19 100.4 ± 3.6 2.67 ± 0.94 299.8 ± 12.5 6 (2) IN8 is a function of A/W This close approximation
25 2.13 ±0.25 196.9 ± 3.5 1.90 ± 0.82 197.8 ± 8.5 7 to that expected for a Nernstian relation between INH
14 2.96 ±0.43 288.9 ±5.9 1.10 ± 0.32 98.8 ± 4.8 16 and A/INB also has been recently demonstrated in
12 3.78 ±0.47 395.2 ±6.0 0.75 ± 0.35 50.5 ± 3.2 13 perfused single heart cell preparations (Brown et al.,
7 4.30 ±0.43 506.3 ±6.2 0.32 ± 0.36 4.7 ± 3.3 13 1981).
All values represent mean ± SEM. Therefore, one of the important consequences of
Values were chosen during onset of action, only, for ouabain, the change in aka induced by digitalis aside from the
and during both onset and washout for AS and actodigin. Not all
values could be obtained for each point, which is why there is such
obvious effects on twitch tension is to alter the up-
a large variation in n, particularly during the rapid washout of the stroke velocity of the cardiac action potential. This
faster-acting agents. effect on Na + current might also explain the shorten-
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2- results allowed a direct correlation of changes in aka
with positive inotropic effects of low concentrations
0
of cardiac steroids.
-2
y= 0.7IX-6I.9 Lee et al. (1980) were the first to use Na-ISE in
r = 0.974
beating fibers in an attempt to provide an answer to
this question. These investigators used periodic stim-
ulation to determine the inotropic effects of various
concentrations of dihydro-ouabain. The positive ino-
tropic effect of this agent was always associated with
-8
a rise in aka- Similarly, aka decreased to its initial
-10
value as twitch tension returned to baseline during
drug washout. Lee and Dagostino (1982) tested several
-12 concentrations of strophanthidin and observed a close
90 85 80 75 70
relation between changes in aka and tension. How-
A / I N a (mV)
ever, the precise temporal relation between changes
in aka and twitch tension could not be derived from
FIGURE 7. Relation between the transmembrane electrochemical these experiments, nor were any other agents tested.
gradient forNa (A/iNa) and the maximum rate of phase 0 depolar- Whereas most investigators assume that the effects of
iation (Vm,). Only the mean changes are presented for clarity of cardiac glycosides derive from the same mechanism,
the illustration. The solid line and the equation represent the there are reports that the therapeutic effects of some
regression line for the relation between percent changes in Vmax
cardiac steroids could be separated from their toxic
and changes in the A/INo- The values of AjiNa were calculated from
the changes in aba, using the value of V at V max of —16 m V obtained
(depolarizing and arrhythmic) effects (Okita et al.,
by Walton and Fozzard (in press). The broken line indicates the 1973; Ten Eick et al., 1973; Mendez et al., 1974).
expected relation if V ma , were a simple function of the electrochem- We have directly investigated these problems in
ical driving force for the Na + inward current (see text for explana- the present series of experiments. We tested the ef-
tion). fects of three different cardiac steroids on the relation
between changes in aka and twitch tension. The tech-
nique of periodically stopping stimulation in order to
ing of the action potential observed with digitalis, measure aka with Na-ISE permits a close correlation
since the plateau of the Purkinje strand action poten- between aka and the twitch in a regularly beating
tial may be maintained in part by Na+ current (Cor- sheep Purkinje fiber (lHz). We found that all concen-
aboeuf et al., 1979). trations of the three agents that produced positive
Discussion inotropy also increased aka- Ouabain was tested only
during the onset of the positive inotropic action, and,
The mechanism underlying the therapeutic action in each case, aka increased immediately prior to or
of digitalis has been uncertain, despite much research. coincident with an increase in the twitch. The other
The most comprehensive explanation relies on the two agents, acetylstrophanthidin and actodigin, have
specific action of glycosides on the Na,K-ATPase. rapid and reversible actions, and we therefore tested
That proposal uses the apparently specific binding of both onset and washout of their positive inotropic
cardiac glycosides to this enzyme system, and the effects. Both agents had exactly the same relation
subsequent inhibition of its ability to maintain the between changes in aka and tension as did ouabain
normal Na and K transmembrane gradients, to explain during the onset of inotropic action, namely, aka
the subcellular basis for digitalis action. This Na changed in parallel with the increase in the twitch.
pump inhibition theory relies entirely on the premise This parallel between aka and tension was also main-
that aka increases, and that it is this rise in aka that tained during washout of positive inotropy. Low con-
ultimately is responsible for the increase in Ca++ centrations of these agents never caused a fall in aka
available to the contractile pool. The increase in Ca++ under all the conditions we used, and when enough
presumably is accomplished via the Na+,Ca++ equi- drug was used to produce a measurable positive ino-
librium exchange mechanisn in heart (Reuter and tropic effect, aka always increased. No Na pump
Seitz, 1968). This explanation predicts a close rela- stimulation was seen at positive inotropic concentra-
tionship between aka and twitch tension throughout tions of any of the three agents; neither was there any
the effect of cardiac glycosides, including concentra- indication of a separation of the increase in aka from
tions that produce only small positive inotropic ef- positive inotropy. We cannot rule out a fall in aka
fects. This is a particularly important point because under all conditions, but we can affirm the relation-
several investigators have reported that low, therapeu- ship between positive inotropic effect and the rise in
tic concentrations of digitalis actually decrease aka via aka-
Na pump stimulation (Blood and Noble, 1978; Deit- The finding that actodigin has the same effects as
mer and Ellis, 1978). Others have suggested that the the other agents further supports the uniform action
anticipated rise in aka might not even be measurable for this family of agents. Actodigin is a semi-synthetic
because of the rapidity of the Na-Ca exchange process cardiac glycoside with its lactone ring attached at the
(Akera and Brody, 1978). Unfortunately, none of these C2 carbon to the steroid nucleus rather than the usual
Downloaded from http://circres.ahajournals.org/ by guest on March 6, 2015704 Circulation Research/VoJ. 52, No. 6, June 1983
C3 attachment as with AS and ouabain (Deghenghi, 1979). This would explain why cardiac glycosides
1970). Several investigators have reported that this characteristically reduce action potential duration
alteration in configuration imparts a greater margin without a decrease in membrane responsiveness (Kas-
of safety (therapeutictoxic) to this type of agent as sebaum, 1963). There is another possibility that might
evaluated in dog heart-lung preparations (Mendez et also contribute to the shortening of the action poten-
al., 1974), isolated dog Purkinje fibers (Gliklich et al., tial. Marban and Tsien (1982) have recently reported
1975), and cat Purkinje-muscle preparations (Wasser- that digitalis increases the slow inward current at
strom and Kabela, unpublished observations). This therapeutic concentrations. The increased Cai entering
would suggest that different cardiac glycosides could by this mechanism could, in turn, increase the Ca-
indeed have selective potencies for a "therapeutic" activated outward current (Isenberg, 1977), causing
receptor which is distinct from a "toxic" receptor, shortening of the action potential. One might there-
presumably the Na,K-ATPase. However, actodigin fore expect an increase in the plateau amplitude or
resembles conventional cardiac glycosides in its duration associated with this phenomenon. We ob-
mechanism of therapeutic action, at least in sheep served only a decrease in plateau voltage and action
Purkinje fibers. potential duration, but others have reported first a
The relation between aka and twitch tension has lengthening then a shortening of Purkinje fiber action
been reported for individual experiments in which 0 potentials (Kassebaum, 1963). This might indicate an
K was used to change aka and twitch in sheep Purkinje increase in Ca++ current through the slow inward
fibers (Eisner et al., 1982) and in dog Purkinje fibers channel which subsequently increases the outward
exposed to digitalis (Lee and Dagostino, 1982). We current, thus shortening the action potential.
report here the relation between aka and twitch ten- Several investigators have recently reported a fall
sion for many experiments (n = 31) using all three in aka at low therapeutic digitalis concentrations (God-
cardiac steroids. Our results agree with these previous fraind and Ghysel-Burton, 1977; Deitmer and Ellis,
reports, namely, that this relation is linear at least in 1978; Ghysel-Burton and Godfraind, 1979). Certain
the range of relatively small increases in twitch force methodological constraints may perhaps have been
(up to about six times). The slope of this relation for present in those experiments. Godfraind and Ghysel-
all three drugs indicates that a 1 ITIM increase in aka Burton used guinea pig atria stimulated with field
causes an increase in force of about 100%. We do not electrodes to demonstrate a fall in Nai and rise in Ki
yet know why this relation appears to be linear. If the in response to nanomolar concentrations of ouabain.
amount of intracellular Ca++ available for contraction However, as Grupp et al. (1982) have recently dem-
is in part determined by the coupling ratio of Na-Ca onstrated, this effect is apparently dependent on the
exchange and the Na+ electrochemical gradient (Sheu involvement of autonomic nerve terminals. Needle
and Fozzard, 1982), then contractile strength is a stimulation of atrial strips rather than whole atria
function of aba as well as aka. Further speculation at failed to produce these ionic changes. It is known that
this time is unwarranted, because we do not yet know digitalis can increase catecholamines release (Seifen,
what other intracellular factors besides aka modulate 1974). Catecholamines have recently been shown to
contraction, nor do we understand fully the relation stimulate the Na pump in cardiac tissues and thus
between aba and twitch tension. reduce aka as measured directly by Na-ISE (Wasser-
Aside from the effects of aNa in twitch tension, strom et al., 1982). Perhaps, then, the site of digitalis
changes in aka have other effects on the electrophys- action to reduce Nai in this case might be to affect Nai
iological functioning of the heart. One of the most and Ki indirectly by stimulation of the Na pump via
prominent of these is the effect of an alteration in the an increased local concentration of catecholamine. In
transmembrane Na+ electrochemical gradient (A/iNa) some of our experiments with low digitalis concentra-
on INB- This would explain the fall in Vmax during tions, propranolol (10~6 M) was added in order to
the action of cardiac glycoside. The fall in Vmax occurs prevent any possible action of endogenous catechol-
without cell depolarization or any significant changes amines (e.g., Fig. 4). This did not alter the relation
in other upstroke characteristics. The relation be- between the rise in aka and twitch tension.
tween the decreases in A/xNa and Vmax roughly ap- Deitmer and Ellis (1978) demonstrated a fall in aka
proximates that expected if Vmax is a function of INH, in some quiescent sheep Purkinje fibers exposed to
as has been reported by several investigators (Brown low therapeutic concentrations of strophanthidin. It is
et al., 1981; Walton and Fozzard, (in press)). Thus a known that stimulation has prominent effects on rate
10% decrease in AftN* causes nearly a 10% fall in Vmax. and magnitude of the inotropic action of digitalis
Deviation of the changes in Vmax from that predicted (Ebner and Reiter, 1977). It is therefore difficult to
simply from the effects of aka change on the Na+ interpret the results of Deitmer and Ellis (1977). In
electrochemical gradient is not surprising, since mul- addition, there are possible explanations for a fall in
tiple factors influence the action potential upstroke. aka other than Na pump stimulation. If digitalis were
For example, the increase in aka may be accompanied to reduce Na+ leak in quiescent fibers, in addition to
by an increase in aba via Na-Ca exchange, secondarily its other actions, this could cause the observed fall in
affecting intercellular resistance. aka at low concentrations. Alternatively, Ferrier (1980)
The fall in A/2Na might also be expected to decrease has suggested that digitalis might reduce the pace-
INa throughout the action potential (Attwell et al., maker current in dog Purkinje fibers. Since this in-
Downloaded from http://circres.ahajournals.org/ by guest on March 6, 2015Wasserstrom et al./ Relation between Nai and Twitch: Digitalis Effects 705
ward current is thought to be carried primarily by atria. Br J Pharmacol 66: 175-184
Na + , (Di Francesco, 1981) it is possible that the small Gliklich JI, Gaffney R, Rosen MR, Hoffman BF (1975) Effects of
AY-22,241 (Actodigin) on electrical and mechanical activity of
decrease in a'na observed by Deitmer and Ellis (1978) cardiac tissues. Eur] Pharmacol 32: 1-9
and by Lee and Dagostino (1982) could come from Godfraind T and Ghysel-Burton J (1977) Binding sites related to
reduction of this inward pacemaker current. ouabain-induced stimulation or inhibition of the sodium pump.
Nature 265: 165-166
Grupp T, Grupp 1L, Ghysel-Burton J, Godfraind T, Schwartz A
(1982) Effects of very low concentrations of ouabain on contrac-
tile force of isolated guinea-pig, rabbit and cat atria and right
We would like to thank Dan Raizen and Tom Thompson for ventricular papillary muscles: An interinstitutional study. J Phar-
their excellent technical assistance, Dorla Wuertemburg for typing macol Exp Ther 220: 145-151
this manuscript, and CO. Lee for helpful discussion. We would Isenberg G (1977) Cardiac Purkinje fibers: [Ca+2]i controls the
also like to thank Drs. W. Simon and D. Ammann for their potassium permeability via the conductance components GK,
generous gift of ETH 227 ion exchange resin. and GK 2 . Pfluegers Arch 371: 77-85
Supported by National Institutes of Health Grants HL-20592 Kassebaum DG (1963) Electrophysiological effects of strophanthin
and HL-07381. in the heart. J Pharmacol Exp Ther 140: 329-338
Dr. Schwartz's present address is: Division of Cardiology, Jewish Langer GA, Serena SD (1970) Effects of strophanthidin upon
Hospital, 216 South Kingshighway Boulevard, St. Louis, Missouri contraction and ionic exchange in rabbit ventricular myocardium.
63110. J Mol Cell Cardiol 1: 65-90
Address for reprints: J.A. Wasserstrom, Department of Medi- Lee CO, Dagostino M (1982) Effect of strophanthidin on intracel-
cine—Box 423, The University of Chicago, 950 East 59th Street, lular Na ion activity and twitch tension on constantly driven
Chicago, Illinois 60637. canine cardiac Purkinje fibers. Biophys J 40: 185-198
Received February 2, 1983; accepted for publication April 21, Lee CO, Kang DH, Sokol JH, Lee KS (1980) Relation between
1983. intracellular sodium ion activity and tension of sheep cardiac
Purkinje fibres exposed to dihydro-ouabain. Biophys J 29: 315-
330
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Ghysel-Burton J, Godfraind T (1979) Stimulation and inhibition of
the sodium pump by cardiotonic steroids in relation to their INDEX TERMS: Intracellular sodium ion activity • Sheep Purkinje
binding sites and their inotropic effect on guinea-pig isolated strands • Cardiac glycosides • Twitch tension
Downloaded from http://circres.ahajournals.org/ by guest on March 6, 2015Relation between intracellular sodium and twitch tension in sheep cardiac Purkinje strands
exposed to cardiac glycosides.
J A Wasserstrom, D J Schwartz and H A Fozzard
Circ Res. 1983;52:697-705
doi: 10.1161/01.RES.52.6.697
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