Download Electrophysiological Actions of Diphenylhydantoin on Rabbit Atria

Electrophysiological Actions
of Diphenylhydantoin on Rabbit Atria
DEPENDENCE ON STIMULATION FREQUENCY,
POTASSIUM, AND SODIUM
By R. A. Jensen, Ph.D., and B. G. Katzung, M.D., Ph.D.
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ABSTRACT
Isolated rabbit left atrial preparations were perfused with Tyrode's solutions
containing 1 to 10 /x,g/ml (4 X 10"6-4 X 10" 5 M) diphenylhydantoin (DPH),
2.6-5.6 niM K + , and 154-308 DIM Na + . Steady-state transmembrane resting
and action potentials were recorded from these preparations with glass microelectrodes at stimulation rates ranging from 0.2 to 3/sec. DPH had little or no
effect on the relationship between extracellular [K + ] and membrane resting
potential. Action potential overshoot was generally decreased by 5 and 10
jug/ml DPH and increased by 1 /xg/ml DPH at stimulation rates of 2 and 3/sec
in the presence of increased [K + ]. DPH and increased [K + ] acted synergistically to shorten action potential duration (measured at 50% repolarization). The
effect of DPH on phase 0 of the action potential (measured as action potential
rise time between 10 and 50% and 50 and 90% depolarization) was markedly
dependent upon drug concentration, extracellular [K + ] and stimulation rate.
The lowest concentration of DPH (1 yxg/ml) usually shortened action potential
rise time, particularly when it had been prolonged by increasing extracellular
[K + ]. Conversely, the highest concentration of DPH (10 jig/ml) and increased
[K + ] acted synergistically to prolong action potential rise time (i.e., decrease
depolarization rate). When present, the depressant effect of DPH on membrane
depolarization was rapidly antagonized by increasing extracellular [Na + ]. It
is proposed that DPH may either enhance or depress (like quinidine) membrane activity in atrial tissue, and that both the direction and magnitude of
effect are strongly dependent upon drug concentration, ionic milieu, and heart
rate.
ADDITIONAL KEY WORDS
antiarrhythmic activity
microelectrode
transmembrane potentials
heart muscle in vitro
• Diphenylhydantoin ( D P H ) has been
shown to be an effective agent in abolishing
various experimental and clinically encountered cardiac arrhythmias (1-5). Although
widespread interest has been shown in DPH,
there remain a number of important questions
regarding its antiarrhythmic actions.
Bigger and associates (6) have reported
that DPH decreases action potential (AP)
From the Department of Pharmacology, University
of California, San Francisco, California 94122.
This investigation was supported in part by
U.S.P.H.S. Grant GM-475 and Bay Area Heart
Research Committee. Dr. Katzung is a Markle Scholar
in Academic Medicine.
Received September 26, 1969. Accepted for
publication November 10, 1969.
Circulation Research. Vol. XXVI, January 1970
duration and increases membrane responsiveness (i.e., dv/dt of phase 0 of the action
potential as a function of membrane potential
preceding the upstroke) in isolated canine
Purkinje fibers. Strauss and co-workers (7)
described similar effects of DPH on membrane responsiveness in rabbit and dog atrial
preparations. Both groups reported that increased membrane responsiveness with DPH
is most noticeable in depressed preparations
(i.e., following toxic concentrations of cardiac
glycosides or cooling or under anoxic conditions). Bigger et al. (6) reported that the
transmembrane potential effects of DPH were
accompanied by improved conduction in
Purkinje fibers. These findings, and other
17
18
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results from both isolated and intact preparations (8-10), contrast substantially with those
previously described for quinidine under
similar conditions (11-13). Quinidine generally prolongs duration of the action potential
and decreases membrane responsiveness and
conductivity in cardiac muscle preparations.
The effects of quinidine on the heart can be
modified by alteration of a number of factors,
including, among others, the concentration of
the drug (14), heart rate (13, 15), and the
extracellular concentration of sodium (16, 17)
and potassium (18-21). We have found (22)
that the effects of DPH on maximum follow
frequency, conduction, sinus nodal rate, and
contractility in isolated rabbit and dog atrial
preparations can be modified by these same
variables. Moreover, from these results we
concluded that DPH is capable of exerting
two opposing effects on the electrical properties of cardiac tissue. That is, under one set of
conditions (e.g., elevated extracellular [K + ],
high stimulation frequencies) DPH exerts a
depressant effect on membrane function similar to that of quinidine, whereas under a
different set of conditions in the same
preparation (e.g., decreased extracellular
[K + ], low stimulation frequencies) DPH may
actually improve membrane electrical activity
relative to controls. In the present investigation we have extended this work to a study of
the effect of several concentrations of DPH on
transmembrane resting and action potentials
in a wide range of potassium and sodium
solutions at various stimulation frequencies.
The data support our previous conclusion
(22) that DPH is capable of exerting
opposing effects on the electrical properties of
cardiac fibers, depending upon drug concentration, ionic environment, and driving rate.
Methods and Materials
Rabbits of either sex (weights 2 to 2.5 kg)
were stunned by a blow to the neck and rapidly
exsanguinated. The heart was removed and the
left atrium dissected free in oxygenated Tyrode's
solution at room temperature. The excised atrium
was trimmed of septal tissue and suspended
horizontally in a 5-ml capacity tissue chamber.
One end of the preparation was impaled on a
strain gauge (Grass FT-03) lever arm. The
JENSEN, KATZUNG
opposite end was fixed to the terminus of an
adjustable rod which provided a means for
establishing and maintaining a constant diastolic
tension (approximately 0.75 g). The temperature
of the tissue chamber was maintained at
36°C ± 0.5°C throughout the experiment.
Rhythmic contractile activity was maintained
by applying slightly supramaximal square wave
pulses of 3-msec duration to the muscle from a
Grass S-4 stimulator and stimulus isolation unit.
Transmembrane potentials were recorded with
flexibly mounted glass microelectrodes filled
with 3M KC1. The resistance of the electrodes
varied from 10 to 30 megohms. Recorded
potentials were led to the input of a high
impedance, neutralized input capacity amplifier
(Winston electronics, S-857). The output of the
S-857 was led to a differential amplifier and
displayed on a dual-beam cathode ray oscilloscope (Tektronix, 565). For voltage calibration a
30 mv signal was introduced between the bath
and ground. Records were photographed using a
Tektronix C-12 oscilloscope camera.
All preparations were perfused by gravity flow
at a rate of approximately 3 ml/min. The
Tyrode solution (control) contained (in M M ) :
NaCl, 154; KC1, 2.2; KH2PO4, 0.4; MgCl,6H.,O,
1.1; NaHCO3, 7.4; CaCL,, 3.0; dextrose, 11.1.
The effects of 1.5 and 10 {JLg/ml diphenylhydantoin sodium (4 X 10"°, 2 X 10"5, 4 X 1 0 - 5 M )
were studied at four different levels of extracellular K+ (2.6, 3.6, 4.6, 5.6 M M ) , and at three
different levels of extracellular Na + (154, 231,
308 MM). Powdered diphenylhydantoin sodium
(Mann Biochemicals) was dissolved directly in
stock solutions of the perfusate shortly before
using. Potassium was added to the solution
reservoir as aliquots of a concentrated KC1
solution made from a Tyrode base.
The general experimental procedure was as
follows: (1) At the outset of each experiment the
tissue was allowed to equilibrate in the control
solution at a basal driving frequency of 1/sec for
at least 60 minutes; (2) following equilibration
the tissue was driven at the basal rate with
periodic (every 15 to 20 minutes) alterations in
rate to lower (0.2/sec) and higher (2/sec and
3/sec) frequencies; (3) steady-state transmembrane potentials were recorded at each driving
frequency in the presence of tlie control and one
or more of the test solutions.
Changes in the upstroke (phase 0) of the
action potential were analyzed in terms of the
time required for the cell to depolarize between
10 and 50% and 50 and 90% of maximum AP
amplitude (RT 10-50, RT 50-90). This made
possible a quantitative expression of differential
drug and ionic effects on slow voltage changes
occurring in the region near the foot and the peak
Circulation Research, Vol. XXVI, January 1970
EFFECT OF DPH ON ATRIAL MEMBRANE POTENTIALS
of the upstroke. The duration of the action
potential was measured at the level of 50%
complete repolarization. Measurements were also
made of the membrane resting potential (E r )
and AP overshoot.
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Results
Experiments were performed on 31 left
atrial preparations. The time required for the
onset (and washout) of the full effect of DPH
was approximately 30 minutes at the basal
driving rate (1/sec). The effect of a change in
extracellular K+ appeared to be complete 10
to 15 minutes following the start of perfusion.
A major problem in any study of transmembrane electrical activity of cardiac muscle is the
relatively large variation of recorded potentials (particularly phase 0 and repolarization
of the action potential) from fiber to fiber in
this type of experiment. In many of our
preparations we found that in spite of the
mechanical activity of the muscle, it was
possible to maintain a satisfactory microelectrode impalement for periods of up to 3 hours
enabling us to analyze the effects of a rather
broad range of drug and ionic variations on
the membrane properties of a single fiber.
Within this time it was usually possible to
perfuse the preparation with at least two
successively higher concentrations of DPH,
including K+ changes at each drug level and
the appropriate controls. In some experiments
we purposely made a number of different
penetrations in different fibers, particularly
19
when DPH and K + -dependent changes in
action potential overshoot and resting potential were being specifically studied and it was
desirable to eliminate any possible errors
arising from amplifier drift.
POTASSIUM-DEPENDENT EFFECTS OF DPH
ON MEMBRANE RESTING AND ACTION POTENTIALS
AT ALTERED STIMULATION FREQUENCIES
Resting Potential
Under drug-free conditions the expected
inverse relationship between resting potential
(E r ) and extracellular [K + ] was observed in
all preparations. Increasing or decreasing the
driving rate had little or no effect on this
relationship. These data are summarized in
Table 1 as control values for DPH response.
DPH, in any concentration, had no significant
effect on the relationship between E r and
extracellular [K + ]. That is, at a given level of
[K + ] (hence E r ), DPH produced no additional change in Er. In a number of
experiments the preparation failed to respond
to electrical stimulation (i.e., propagated
action potentials were abolished) when it was
driven at the highest stimulation frequency
(3/sec) in the presence of a Tyrode solution
containing 10 //.g/ml DPH and 5.6 mM K + .
However, it was always possible to record a
relatively stable E r of approximately 75 to 77
mv (Table 1) under these conditions.
Action Potential Overshoot
The effect of DPH (1, 10 /ig/ml) on action
potential overshoot in altered K+ solutions is
TABLE 1
Potassium-Dependent Effect of DPH (10 ng/ml) on Membrane Resting Potential (mv)
K + (mM) DlPHCsr/ml)
0.2/sec
10
89.7 ± 0.7
89.4 ± 1.1
10
87.4 ± 1.0
87.6 ± 1.5
2.6
3.6
4.6
10
5.6
10
84.2
84.5
78.2
77.1
±1.9
± 2.0
± 2.1
± 1.5
Stimulation frequency
1/sec
2/sec
3/sec
1.0
1.6
1.3
1.0
89.4 ± 0.8
88.9 ± 0.9
88.9 ± 1 . 0
88.2 ± 2.0
87.8 ± 1.1
86.9 ± 2.0
87.9 ± 1.5
86.4 ± 1 . 2
84.2 ± 1.3
84.3 ± 1.6
84.7 ± 1.1
83.3 ± 2.3
83.9 ± 2.1
83.1 ± 2.0
78.3 ± 3.1
77.4 ± 2.00
77.4 ± 3.2
76.2 ± 2.4
76.9 ± 2.4
76.5 ± 3.3
89.6 ±
89.5 ±
. 88.2 ±
87.1 ±
Mean values ± SE recorded from 5 preparations (minimum of 13 and maximum of 38 observations at each point).
Circulation Research, Vol. XXVI,
January 1970
JENSEN, KATZUNG
20
99'p
Action Potential Rise Time
t, l_JJg/ml DPB'
|j
H
?—
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0.2
'^lOug/miDPH !
1
2
Stimulation Frequency (cps)
FIGURE 1
Potassium-dependent effect of 1, 10 ^g/ml (4 x 10-",
4 X 1O~B M) DPH on action potential (AP) overshoot
at altered stimulation frequencies (0.2-3/sec). DF =
drug free. Extracellular [K + ] = 2.6 m« (dashed lines)
and 5.6 mM (solid lines). Mean values ± SE recorded
from 5 rabbit left atrial preparations (minimum of 18
and maximum of 23 observations at each point).
Tyrode's solution; 36° C ± 0.5°C.
graphically illustrated in Figure 1. Mean
values ± SE are shown for a minimum of 18
and a maximum of 23 observations at each
point. Under drug-free conditions, increasing
extracellular [K + ] produced a progressive
decrease in the magnitude of overshoot. The
lowest concentration of DPH (1 /Ltg/ml) had
little effect on K+-dependent changes in
overshoot except at 4.6 and 5.6 mM K + , where
sometimes it reversed the depression that
resulted in the increased [K + ]. The highest
concentration of DPH (10 jug/ml) had little
effect on overshoot in 2.6 mM K+ Tyrode's
solution (Fig. 1), but substantially decreased
it when extracellular [K + ] was raised to 4.6 or
5.6 mM (particularly at higher driving rates).
Following Weidmann's initial study (23)
using Purkinje fibers, it has been shown in
various cardiac tissue (24) that, within certain
limits, the maximum rate of rise of phase 0 of
the action potential is related to the membrane potential from which the action potential arises. In the present investigation with
rabbit atria, the effects of DPH on phase 0 of
the action potential (both quantitative and
qualitative) depended greatly on extracellular
[K + ] (primarily, it appears, through changes
in E r ), drug concentration, and stimulation
rate. Representative records are illustrated in
Figures 2 and 3 (high sweep speed records),
which show superimposed tracings of action
potentials recorded, in each case, from a single
fiber in various extracellular K+ solutions
before and during perfusion with DPH. In
Figure 2 the rate of rise of the action potential
is increased over the drug-free value by 1
jiig/ml DPH at stimulation frequencies of 2
and 3/sec in 5.6 mM K+ Tyrode's solution. By
contrast the same concentration of DPH
exerted no visible effect on the action
potential upstroke in 2.6 mM Tyrode's solution, regardless of frequency. In the experiment illustrated in Figure 3, 10 jug/ml DPH
exerted an obvious depressant effect on
membrane depolarization in both 3.6 and 4.6
mM K+ Tyrode's solution. It C ± 0.5° C .
this effect varies substantially with both the
extracellular [K + ] and stimulation rate.
When viewed at high sweep speeds, the
upstroke of the action potential consists
roughly of three segments of voltage changes
with time: an initial slow foot, a rapid, almost
linear phase, and a slowly curving terminal
phase. Measurements of action potential rise
time of 10 to 50% (RT 10-50), and 50 to 90%
(RT 50-90) depolarization provided a means
of determining if DPH exerted differential
effects on slow voltage changes at the foot and
the peak of the action potential. Potassium
and frequency-dependent effects of DPH on
RT 10-50 and RT 50-90 recorded from 24
atrial preparations are summarized in Figure
4. The lowest concentration of DPH (1
/xg/ml) has little effect on either segment in
Circulation Research, Vol. XXVI, January 1970
21
EFFECT OF DPH ON ATRIAL MEMBRANE POTENTIALS
2.6 mM K' - 1 ug/ml DPH
0.2/sec
2/sec
2/sec
3/sec
5.6 mM K - I jjg/ml
yg/rr DPH
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0.2/sec
3/sec
l/sec
FIGURE 2
Potassium-dependent effect of 1 iig/ml (4 X 10~6M) DPH on the action potential (AP) upstroke
(fast sweep speed record) and repolarization (slow sweep speed record) at altered stimulation
frequencies (0.2-3/sec). Rabbit left atrium. 36°C. Superimposed tracings of action potentials
recorded from a single atrial fiber before and during perfusion with DPH in 2.6 THM K +
Tyrode's solution (top), and before and during perfusion with DPH in 5.6 TUM K+ Tyrode's
solution (bottom). DF = drug free.
2.6 and 3.6 HIM K+ but tends to shorten each
(i.e., decreases rise time) in 4.6 and 5.6 mM
K+, particularly the latter. The effects of 10
/xg/ml, and in many cases that of 5 fig/ml, are
generally similar to those expected with
quinidine under comparable conditions. In 4.6
and 5.6 mM K+ Tyrode's solution it is
particularly evident (Fig. 4) that both RT 1050 and RT 50-90 are prolonged by 10 fig/ml
DPH. Changes in RT 10-50 appear to be
slightly greater than those in RT 50-90.
Action
Potential Duration
Representative records of potassium and
frequency-dependent effects of DPH on the
repolarization phase of action potentials recorded from two atrial fibers are shown in the
slow sweep speed tracings in Figures 2 and 3.
Similar effects of DPH on action potential
duration measured at 50% repolarization
are quantitatively summarized in Table 2.
Changes in duration of the action potential
Circulation Research, Vol. XXVI, January 1970
were determined in both control and test
solutions in 26 preparations. In the experiments presented in Figures 2 and 3, this
duration is either unchanged or shortened in
the presence of DPH, depending (as did
action potential overshoot and rise time) on
drug concentration, extracellular [K + ], and
stimulation frequency. In the range of concentrations studied, both DPH and increased
[K + ] (separately or in combination) usually
shortened duration of the action potential at
stimulation rates of 1-3/sec. In addition, the
magnitude of shortening produced by one
depended critically on the concentration of
the other. In the presence of 2.6 mM K + , DPH
always shortened duration of the action
potential with respect to drug-free values,
except at the lowest stimulation frequency
(Table 2). By contrast, the drug had little or
no effect on duration of the action potential in
5.6 mM K+ Tyrode's solution, when the action
potential duration was diminished by aug-
JENSEN, KATZUNG
22
3.6 mM K + - 10 Mfl/ml DPH
of,
0.2/sec
2/sec
3/s.
2/sec
3/sec
4.6 mM K + - lOug/ml DPH
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0.2/sec
i/sec
FIGURE 3
Potassium-dependent effect of 10 ng/ml (4 XlO~5 M) DPH on the action potential (AP) upstroke
(fast sweep speed record) and repolarization (slow sweep speed record) at altered stimulation
frequencies (0.2-3/sec). Rabbit left atrium. 36°C. Superimposed tracings of action potentials
recorded from a single atrial fiber before and during perfusion with DPH in 3.6 mM K +
Tyrode's solution (top), and before and during perfusion with DPH in 4.6 mat K + Tyrode's
solution (bottom). DF = drug free.
TABLE 2
Potassium-Dependent Effect of DPH on Action Potential Duration (msec)
K+
(mM)
DFH
(»»g/ml)
0.2/sec
Stimulation frequency
l/sec
l/sec
2/sec
± 1.0
± 0.4
± 0.3
± 0.7
± 0.1
± 0.2
± 0.3
± 0.4
25.1 ±0.1
20.6 ±0.7
18.0 ± 1.4
18.4 ± 1.4
33.7
28.5
28.4
28.1
± 1.5
± 1.5
±3.1
± 2.1
33.9
26.4
26.0
26.7
± 1.5
± 0.9
± 3.7
=>= 2.9
1
5
10
7.5
7.5
7.0
7.1
8.2
8.0
7.9
8.2
22.5 ±
10.6 ±
18.0 ±
18.7 ±
1.6
1.3
1.3
2.4
29.9
27.4
28.0
28.6
±
±
±
±
1.6
1.9
1.1
2.0
29.3
27.2
26.7
27.0
±
±
±
±
1.5
1.3
0.6
2.0
1
5
10
7.5
7.9
7.0
6.8
± 0.1
± 0.6
± 0.3
± 0.7
18.1 ±
17.7 ±
17.5 ±
17.2 ±
1.2
1.3
1.5
1.7
26.3
26.2
25.8
24.8
±
±
±
±
1.5
1.6
1.3
1.7
27.6
26.0
26.5
27.5
±
±
±
±
1.5
1.3
0.1
1.6
1
5
10
6.2
6.0
5.9
6.1
± 0.3
± 0.2
± 0.3
± 0.7
16.3 ±0.6
15.7 ± 1.6
16.4 ± 1.6
17.0 ± 1.4
25.4
25.7
26.1
26.1
±
±
±
±
1.2
1.2
1.6
2.8
26.6
25.9
25.5
27.8
±
±
±
±
1.5
2.4
2.2
3.4
2.6
1
5
10
3.6
4.6
5.6
Mean values ± SB recorded from 26 preparations (minimum of 19 and maximum of 31 observations at each point).
Circulation Research, Vol. XXVI, January 1970
23
EFFECT OF DPH ON ATRIAL MEMBRANE POTENTIALS
8
6
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LU
5
a.
FIGURE 4
Potassium-dependent effect of DPH on action potential rise time between 10 and 50% (RT
10-50) and 50 and 90% (RT 50-90) depolarization at stimulation frequencies of 0.2/sec (top
left), 1/sec (top right), 2/sec (bottom left), and 3/sec (bottom right). Means ± SE compiled
from experiments on 24 rabbit left atrial preparations (minimum of 31 and maximum of 69
observations at each point). *P > 0.05 (both RT 10-50 and RT 50-90); i P > 0.05 (RT 50-90 but
not RT 10-50). Each bar has two components: 10-50% and 50-90% rise time.
mented potassium. In some fibers, duration of
action potential was slightly prolonged by
DPH rather than shortened in 5.6 HIM K +
Tyrode's solution. These effects did not appear
to be significant.
In several preliminary experiments we
found that when DPH is administered in the
commercial diluent supplied for parenteral use
(propylene glycol, 40%; ethanol, 10%, in
water) duration of the action potential is
substantially increased rather than decreased
in the highest K+ solutions, and only slightly
increased or unchanged in the lowest K+
solutions. Bigger et al. (6) reported that the
Circulation Research, Vol. XXVI, January 1970
commercial diluent diminished DPH-induced
shortening of the action potential duration in
canine Purkinje fibers. These and other results
(10) leave little doubt that the diluent per se
exerts pharmacologic effects, which, at least in
isolated tissue studies, may obscure true DPH
response.
SODIUM REVERSAL OF THE EFFECT OF DPH
ON TRANSMEMBRANE ACTION POTENTIALS
It is known that increasing the extracellular
sodium concentration will diminish or reverse
some of the depressant effects of quinidine on
electrical properties of cardiac tissue (16, 17).
In five experiments we investigated the
24
JENSEN, KATZUNG
5.6 mM K + - 10pg/ml DPH
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1/sec
0.2/sec
50
mV
2/sec
3/sec
FIGURE 5
Reversal of effect of 10 ng/ ml (4 X 1O~5 M) DPH in 5.6 mM K+ Tyrode's solution by increasing
extracellular [Na + ] (154-308 min). Superimposed tracings of action potentials recorded from
single cell before and during perfusion with increased [Na + ], DPH concentration maintained
constant. Rabbit left atrium; 36°C; stimulation frequency = 0.2-3/sec.
relationship between extracellular [Na + ] and
DPH-induced changes in rabbit atrial transmembrane potentials. Typical results are
illustrated in Figure 5. In this experiment a
100% increase in extracellular [Na + ] (154 to
308 mM Na + ) significantly antagonized the
membrane effects of 10 /ng/ml DPH in 5.6 mM
K+ Tyrode's solution. Similar but less marked
changes were produced by increasing extracellular [Na + ] by 50%. The observed changes in
phase 0 and the overshoot of the action
potential with increased Na + undoubtedly
account for the sodium reversal of a depressant effect of DPH on conduction velocity
which we reported in a previous study (22).
Discussion
These results show that DPH is capable of
exerting a wide range of effects on transmembrane electrical properties of isolated rabbit
atria, including a quinidine-like depression of
the depolarization phase of the action potential, an enhancement of the depolarization
phase (i.e., an increase in the rate of
Circulation Research, Vol. XXVI, January 1970
EFFECT OF DPH ON ATRIAL MEMBRANE POTENTIALS
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depolarization), depression or enhancement
of the action potential overshoot, and increased rate of repolarization of the action
potential. Both the direction and the magnitude of DPH effects on these properties were
found to depend primarily upon drug concentration. Also, the observed effects were markedly sensitive to even small changes in
extracellular [ K+ ] or the driving frequency.
Finally, we have shown that the depressant
effect of DPH on membrane depolarization,
like that of quinidine, may be antagonized by
increasing extracellular [Na + ].
A major difficulty encountered in any invitro study of drug action is deciding what
concentrations of the drug in the isolated
tissue chamber correspond to therapeutic and
toxic levels of the drug in man. This question
is particularly important in the interpretation
of the present results in view of the substantial
variation in response (qualitative and quantitative) with DPH concentration. The dose
range used in this study was 1-10 /^g/ml
(4 X 10-0-4 X 10- 5 M) DPH. On the basis of
data available in the literature (5, 22) it appears that during the period when DPH is
exerting its antiarrhythmic effects in animals
and man the plasma concentration of the drug
is in the range of 5-25 /ng/ml (2xlO" 5 1 X K H M ) and is probably no lower than 1
/^g/ml ( 4 X 1 0 - ° M ) . Also, it has been established by Zeft et al. (25) that during the first
few hours after intravenous administration of
a single dose of DPH to pigs, the amount of
the drug concentrated in myocardial tissue is
in reasonable equilibrium with that located in
the blood. In view of these findings we feel
that the concentrations we used (1, 5, and 10
/xg/ml bath solution) are at least reasonably
close to the concentrations achieved in the invivo application of DPH.
The results presented in Figures 2 through 4
emphasize the importance of DPH concentration as a variable in these studies. In the
presence of the lowest concentration (1
/i,g/ml) of DPH and elevated extracellular
[K + ] (4.6, 5.6 HIM) depolarization rate was
noticeably increased with respect to drug-free
values with little or no change in resting
Circulation Research, Vol. XXVI, January 1970
25
potentials, indicating that membrane responsiveness was increased under these circumstances. By contrast, depolarization rate was
always decreased by the highest concentration
(10 /xg/ml) and usually by the intermediate
concentration (5 /xg/ml) under comparable
conditions, indicating a decrease in membrane
responsiveness. Bigger and associates (6) and
Strauss and co-workers (7) have demonstrated that DPH in a range of 10" 8 -10- 5 M
(.0025-2.5 //.g/ml) increases membrane responsiveness in canine Purkinje fibers (6) and
rabbit and canine atrial fibers (7), particularly
in preparations that have previously been
depressed by toxic concentrations of the
cardiac glycosides, or cooling, or anoxia. In
the present study with rabbit atria, depression
of membrane depolarization occurred in the
presence of DPH concentrations (2 X 10"5,
4 X 10~ 8 M) that, in comparison, might be
considered excessive for antiarrhythmic response. However, even if we assume that this
is correct, these results are no less significant
for it is still possible, and indeed pertinent, to
consider that depression of membrane responsiveness by DPH represents an important
toxic manifestation of the drug—particularly
in patients with altered plasma K+ levels. The
extracellular [K + ] concentrations utilized in
this study varied from 2.6-5.6 mM. Both
Bigger et al. (6), and Strauss et al. (7) used
solutions containing 3.0 mM K + , which is
below the potassium levels at which we
usually saw depression of membrane function
by DPH, and somewhat less than the reported
physiological range of 5.0-5.5 mM (26).
A synergistic relationship between extracellular [K + ] and the cardiac effects of quinidine
has been documented by a number of
investigators. For example, both Holland (27)
and Armitage (28) found that the depressant
effects of quinidine on contractile force and
spontaneous rate of isolated rabbit atria were
blocked by lowering extracellular [K + ]. Recently Watanabe and Dreifus (20) reported
that prolongation of intra-atrial, A-V nodal,
and His-Purkinje conduction time by quinidine in isolated rabbit atria was antagonized in
low extracellular [K + ] and enhanced in high
26
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extracellular [K + ]. Watanabe and associates
(19) had previously reported similar results in
ventricular preparations. Moreover, they correlated extracellular and transmembrane electrical phenomena by showing that depression
of conduction and the maximal rate of
depolarization by quinidine are simultaneously reversed by lowering extracellular [K + ].
Our results, both the present ones and those
recently reported (22), indicate that the
depressant action of DPH on membrane
function is dependent upon extracellular [K + ]
in a manner which is similar if not identical to
that of quinidine. Indeed it appears that,
given the right conditions, a common property
of DPH and quinidine is depression of the
rate of depolarization of cardiac cells and that
this property is antagonized by low K+ and
enhanced by high K+ in the surrounding
medium.
Additional evidence indicating that DPH
and quinidine may exert depressant effects on
depolarization mechanisms via a common
pathway is presented in Figure 5. In this
experiment a reversal of the depressant effect
of DPH on atrial cell depolarization was
rapidly accomplished by increasing the level
of NaCl in the perfusate in spite of the
continued presence of DPH in the solution. A
similar reversal of the depressant effects of
quinidine on rabbit atria by various sodium
salts (lactate, sulfate, chloride) has been
described by Cox and West (16) who
concluded that reversal resulted from a
specific effect of Na + , rather than the anions
utilized or a change in the osmolarity of the
solution. Examination of their records and our
own shows close similarities. The increase in
depolarization rate in elevated [Na + ] is
greater than one would expect to result from
increased resting potential, therefore it would
be anticipated that an increase in external
[Na + ] would exert a favorable effect on
depolarization and conduction, as we have
previously shown (22), by increasing the Na +
gradient.
We can only speculate on the importance of
the relationship between DPH and K + , and
that between DPH and Na + , at the present
JENSEN, KATZUNG
time. More definite conclusions on the role
played by these.ions in therapeutic and toxic
response to DPH must await detailed electrophysiological studies of K + - and Na +-dependent drug effects on refractoriness, automaticity, and conduction in various cardiac tissues.
We can conclude, however, that the effect of
DPH on atrial transmembrane potentials and
conductivity is complex, and depends upon a
somewhat delicate balance between drug
concentration, heart rate, and extracellular
sodium and potassium.
Acknowledgment
We wish to thank Miss Margaret J. Ballage for her
interest and assistance in these studies.
References
1.
HARRIS, S., AND KOKERNOT, R.
H.:
Effects of
diphenylhydantoin sodium (dilantin sodium)
upon ectopic ventricular tachycardia in acute
myocardial infarction. Amer J Physiol 163:
505, 1950.
2.
SCHERF, D., BLUMENFELD, S., TANER, D., AND
TILDIZ, M.: Effect of diphenylhydantoin sodium on atrial flutter and fibrillation provoked by
focal application of aconitine or delphenine.
Amer Heart J 60: 937, 1960.
3. LEONARD, W. A.:
Use of
diphenylhydantoin
(Dilatin) sodium in the treatment of ventricular tachycardia. Arch Intern Med 101: 714,
1958.
4.
SANO, T., SUZUKI, F., SATO, S., AND IIDA,
Y.:
Mode of action of new antiarrhythmic agents.
Jap Heart J 9: 161, 1968.
5.
BIGGER, J. T., JR., SCHMIDT, D. H., AND KUTT,
H.: Relationship between the plasma level of
diphenylhydantoin in sodium and its cardiac
antiarrhythmic effects. Circulation 38: 363,
1968.
6.
BIGGER,
J.
T.,
JR.,
BASSETT,
A.
L.,
AND
HOFFMAN, B. F.: Electrophysiological effects
of diphenylhydantoin on canine Purkinje
fibers. Circ Res 22: 221, 1968.
7.
STRAUSS, H. C , BIGGER, J. T., JR., BASSETT, A.
L., AND HOFFMAN, B. F.: Actions of diphenylhydantoin on the electrical properties of
isolated rabbit and canine atria. Circ Res 23:
463, 1968.
8.
HELFANT, R. H., SCHERLAG, B. J., AND DAMATO,
A. N.: Electrophysiological properties of diphenylhydantoin sodium as compared to procaine amide in the normal and digitalisintoxicated heart. Circulation 36: 108, 1967.
9.
ROSATI, R. A., ALEXANDER, J. A., SCHAAL, S. F.,
AND WALLACE, A. G.: Influence of diphenylCirculation Research, Vol. XXVI, January 1970
27
EFFECT OF DPH ON ATRIAL MEMBRANE POTENTIALS
hydantoin on electrophysiological properties of
the canine heart. Circ Res 21: 757, 1967.
10.
19.
Electrophysiological antagonism and synergism of potassium and antiarrhythmic agents.
Amer J Cardiol 12: 702, 1963.
SASYNIUK, B. I., AND DRESEL, P. E.: Effect of
diphenylhydantoin on conduction in isolated,
blood-perfused dog hearts. J Pharmacol Exp
Ther 161: 191, 1968.
11. WEIDMAN, S.: Effects of calcium ions and local
anaesthetics on electrical properties of Purkinje
fibers. J Physiol (London) 129: 568, 1955.
20.
13. WEST, T. C , AND AMORY, D. W.: Single fiber
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
recording of the effects of quinidine at atrial
and pacemaker sites in the isolated right atrium
of the rabbit. J Pharmacol Exp Ther 130: 183,
1960.
14.
SOKOLOW,
M.,
AND BALL,
R.
E.:
Factors
influencing conversion of chronic atrial fibrillation with special reference to serum quinidine
concentration. Circulation 14: 568, 1956.
15. JOHNSON,
E.
A.,
AND MCKINNON,
M.
21.
17.
KENNEDY,
B. L., AND WEST, T.
C:
Factors
influencing quinidine-induced changes in excitability and contractility. J Pharmacol Exp Ther
168: 47, 1969.
18. LEE, Y.: Quinidine intoxication: Experimental
study of the effect of molar sodium lactate and
potassium chloride. Amer Heart J 60: 785,
1960.
Circulation Research, Vol. XXVI, January 1970
BRANDFONBRENNER,
M.,
KRONHOLM,
J., AND
JONES, H. R.: Effect of serum potassium
concentration on quinidine toxicity. J Pharmacol Exp Ther 154: 250, 1966.
22.
KATZUNG, B. G., AND JENSEN, R. A.: Depressant
action of diphenylhydantoin on electrical and
mechanical properties of isolated rabbit and
dog atria—dependence on sodium and potassium. Amer Heart J, in press.
23. WEIDMANN, S.: Effect of the cardiac membrane
potential on the rapid availability of the
sodium carrying systems. J Physiol (London)
127: 213, 1955.
24.
HOFFMAN,
B.
F.,
AND CRANEFIELD,
P.
F.:
Electrophysiology of the Heart. New York,
McGraw-Hill Book Co., 1960.
G.:
Differential effect of quinidine and pyrilamine
on the myocardial action potential at various
rates of stimulation. J Pharmacol Exp Ther
120: 460, 1957.
16. Cox, A. R., AND WEST, T. C : Sodium lactate
reversal of quinidine effect studied in rabbit
atria by the microelectrode technique. J
Pharmacol Exp Ther 131: 212, 1961.
WATANABE, Y., AND DREIFUS, L. S.: Interactions
of quinidine and potassium on atrioventricular
transmission. Circ Res 20: 434, 1967.
12. VAUGHAN WILLIAMS, E. M.: Mode of action of
quinidine on isolated rabbit atria interpreted
from intracellular potential records. Brit J
Pharmacol 13: 276, 1958.
WATANABE, Y., DREIFUS, L. S., AND LIKOFF, W.:
25.
ZEFT, H. J., WHALEN, R. E., RATLIFF, N. B., JR.,
DAVENPORT, R. D., JR., AND MCINTOSH, H. D.:
Diphenylhydantoin therapy in experimental
myocardial infarction. J Pharmacol Exp Ther
162: 936, 1960.
26. SPECTOR, W M . S., (ed.): Handbook of Biological
Data. Philadelphia, W. R. Saunders Co., 1956,
p. 53.
27. HOLLAND, W. C : A possible mechanism of action
of quinidine. Amer J Physiol 190: 492,
1957.
28. ARMITAGE, A. K.: Influence of potassium
concentration on the action of quinidine and of
some antimalarial substances on cardiac muscle. Brit J Pharmacol 12: 74, 1957.
Electrophysiological Actions of Diphenylhydantoin on Rabbit Atria: DEPENDENCE ON
STIMULATION FREQUENCY, POTASSIUM, AND SODIUM
R. A. JENSEN and B. G. KATZUNG
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Circ Res. 1970;26:17-27
doi: 10.1161/01.RES.26.1.17
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