Electrophysiological Actions of Diphenylhydantoi on …circres.ahajournals.org/content/circresaha/26/1/17.full.pdf · Electrophysiological Actions of Diphenylhydantoi on Rabbin t

Electrophysiological Actionsof 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.

ABSTRACTIsolated rabbit left atrial preparations were perfused with Tyrode's solutions

containing 1 to 10 /x,g/ml (4 X 10"6-4 X 10"5M) diphenylhydantoin (DPH),2.6-5.6 niM K + , and 154-308 DIM Na + . Steady-state transmembrane restingand action potentials were recorded from these preparations with glass micro-electrodes at stimulation rates ranging from 0.2 to 3/sec. DPH had little or noeffect on the relationship between extracellular [K + ] and membrane restingpotential. Action potential overshoot was generally decreased by 5 and 10jug/ml DPH and increased by 1 /xg/ml DPH at stimulation rates of 2 and 3/secin the presence of increased [K + ]. DPH and increased [K + ] acted synergistical-ly to shorten action potential duration (measured at 50% repolarization). Theeffect of DPH on phase 0 of the action potential (measured as action potentialrise time between 10 and 50% and 50 and 90% depolarization) was markedlydependent upon drug concentration, extracellular [K + ] and stimulation rate.The lowest concentration of DPH (1 yxg/ml) usually shortened action potentialrise 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., decreasedepolarization rate). When present, the depressant effect of DPH on membranedepolarization was rapidly antagonized by increasing extracellular [Na+]. Itis proposed that DPH may either enhance or depress (like quinidine) mem-brane activity in atrial tissue, and that both the direction and magnitude ofeffect are strongly dependent upon drug concentration, ionic milieu, and heartrate.

ADDITIONAL KEY WORDS antiarrhythmic activitytransmembrane potentials heart muscle in vitro microelectrode

• Diphenylhydantoin (DPH) has beenshown to be an effective agent in abolishingvarious experimental and clinically encoun-tered cardiac arrhythmias (1-5). Althoughwidespread interest has been shown in DPH,there remain a number of important questionsregarding its antiarrhythmic actions.

Bigger and associates (6) have reportedthat DPH decreases action potential (AP)

From the Department of Pharmacology, Universityof California, San Francisco, California 94122.

This investigation was supported in part byU.S.P.H.S. Grant GM-475 and Bay Area HeartResearch Committee. Dr. Katzung is a Markle Scholarin Academic Medicine.

Received September 26, 1969. Accepted forpublication November 10, 1969.

duration and increases membrane responsive-ness (i.e., dv/dt of phase 0 of the actionpotential as a function of membrane potentialpreceding the upstroke) in isolated caninePurkinje fibers. Strauss and co-workers (7)described similar effects of DPH on mem-brane responsiveness in rabbit and dog atrialpreparations. Both groups reported that in-creased membrane responsiveness with DPHis most noticeable in depressed preparations(i.e., following toxic concentrations of cardiacglycosides or cooling or under anoxic condi-tions). Bigger et al. (6) reported that thetransmembrane potential effects of DPH wereaccompanied by improved conduction inPurkinje fibers. These findings, and other

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18 JENSEN, KATZUNG

results from both isolated and intact prepara-tions (8-10), contrast substantially with thosepreviously described for quinidine undersimilar conditions (11-13). Quinidine general-ly prolongs duration of the action potentialand decreases membrane responsiveness andconductivity in cardiac muscle preparations.

The effects of quinidine on the heart can bemodified by alteration of a number of factors,including, among others, the concentration ofthe drug (14), heart rate (13, 15), and theextracellular concentration of sodium (16, 17)and potassium (18-21). We have found (22)that the effects of DPH on maximum followfrequency, conduction, sinus nodal rate, andcontractility in isolated rabbit and dog atrialpreparations can be modified by these samevariables. Moreover, from these results weconcluded that DPH is capable of exertingtwo opposing effects on the electrical proper-ties of cardiac tissue. That is, under one set ofconditions (e.g., elevated extracellular [K+],high stimulation frequencies) DPH exerts adepressant effect on membrane function simi-lar to that of quinidine, whereas under adifferent set of conditions in the samepreparation (e.g., decreased extracellular[K+], low stimulation frequencies) DPH mayactually improve membrane electrical activityrelative to controls. In the present investiga-tion we have extended this work to a study ofthe effect of several concentrations of DPH ontransmembrane resting and action potentialsin a wide range of potassium and sodiumsolutions at various stimulation frequencies.The data support our previous conclusion(22) that DPH is capable of exertingopposing effects on the electrical properties ofcardiac fibers, depending upon drug concen-tration, ionic environment, and driving rate.

Methods and MaterialsRabbits of either sex (weights 2 to 2.5 kg)

were stunned by a blow to the neck and rapidlyexsanguinated. The heart was removed and theleft atrium dissected free in oxygenated Tyrode'ssolution at room temperature. The excised atriumwas trimmed of septal tissue and suspendedhorizontally in a 5-ml capacity tissue chamber.One end of the preparation was impaled on astrain gauge (Grass FT-03) lever arm. The

opposite end was fixed to the terminus of anadjustable rod which provided a means forestablishing and maintaining a constant diastolictension (approximately 0.75 g). The temperatureof the tissue chamber was maintained at36°C ± 0.5°C throughout the experiment.

Rhythmic contractile activity was maintainedby applying slightly supramaximal square wavepulses of 3-msec duration to the muscle from aGrass S-4 stimulator and stimulus isolation unit.Transmembrane potentials were recorded withflexibly mounted glass microelectrodes filledwith 3M KC1. The resistance of the electrodesvaried from 10 to 30 megohms. Recordedpotentials were led to the input of a highimpedance, neutralized input capacity amplifier(Winston electronics, S-857). The output of theS-857 was led to a differential amplifier anddisplayed on a dual-beam cathode ray oscillo-scope (Tektronix, 565). For voltage calibration a30 mv signal was introduced between the bathand ground. Records were photographed using aTektronix C-12 oscilloscope camera.

All preparations were perfused by gravity flowat a rate of approximately 3 ml/min. TheTyrode 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 diphenylhydan-toin sodium (4 X 10"°, 2 X 10"5, 4 X 10- 5 M)

were studied at four different levels of extracellu-lar K+ (2.6, 3.6, 4.6, 5.6 M M ) , and at threedifferent levels of extracellular Na+ (154, 231,308 MM). Powdered diphenylhydantoin sodium(Mann Biochemicals) was dissolved directly instock solutions of the perfusate shortly beforeusing. Potassium was added to the solutionreservoir as aliquots of a concentrated KC1solution made from a Tyrode base.

The general experimental procedure was asfollows: (1) At the outset of each experiment thetissue was allowed to equilibrate in the controlsolution at a basal driving frequency of 1/sec forat least 60 minutes; (2) following equilibrationthe tissue was driven at the basal rate withperiodic (every 15 to 20 minutes) alterations inrate to lower (0.2/sec) and higher (2/sec and3/sec) frequencies; (3) steady-state transmem-brane potentials were recorded at each drivingfrequency in the presence of tlie control and oneor more of the test solutions.

Changes in the upstroke (phase 0) of theaction potential were analyzed in terms of thetime required for the cell to depolarize between10 and 50% and 50 and 90% of maximum APamplitude (RT 10-50, RT 50-90). This madepossible a quantitative expression of differentialdrug and ionic effects on slow voltage changesoccurring in the region near the foot and the peak

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EFFECT OF DPH ON ATRIAL MEMBRANE POTENTIALS 19

of the upstroke. The duration of the actionpotential was measured at the level of 50%complete repolarization. Measurements were alsomade of the membrane resting potential (Er)and AP overshoot.

ResultsExperiments were performed on 31 left

atrial preparations. The time required for theonset (and washout) of the full effect of DPHwas approximately 30 minutes at the basaldriving rate (1/sec). The effect of a change inextracellular K+ appeared to be complete 10to 15 minutes following the start of perfusion.A major problem in any study of transmem-brane electrical activity of cardiac muscle is therelatively large variation of recorded poten-tials (particularly phase 0 and repolarizationof the action potential) from fiber to fiber inthis type of experiment. In many of ourpreparations we found that in spite of themechanical activity of the muscle, it waspossible to maintain a satisfactory microelec-trode impalement for periods of up to 3 hoursenabling us to analyze the effects of a ratherbroad range of drug and ionic variations onthe membrane properties of a single fiber.Within this time it was usually possible toperfuse the preparation with at least twosuccessively higher concentrations of DPH,including K+ changes at each drug level andthe appropriate controls. In some experimentswe purposely made a number of differentpenetrations in different fibers, particularly

when DPH and K + -dependent changes inaction potential overshoot and resting poten-tial were being specifically studied and it wasdesirable to eliminate any possible errorsarising from amplifier drift.

POTASSIUM-DEPENDENT EFFECTS OF DPHON MEMBRANE RESTING AND ACTION POTENTIALSAT ALTERED STIMULATION FREQUENCIESResting Potential

Under drug-free conditions the expectedinverse relationship between resting potential(Er) and extracellular [K + ] was observed inall preparations. Increasing or decreasing thedriving rate had little or no effect on thisrelationship. These data are summarized inTable 1 as control values for DPH response.DPH, in any concentration, had no significanteffect on the relationship between Er andextracellular [K+]. That is, at a given level of[K+] (hence Er), DPH produced no addi-tional change in Er. In a number ofexperiments the preparation failed to respondto electrical stimulation (i.e., propagatedaction potentials were abolished) when it wasdriven at the highest stimulation frequency(3/sec) in the presence of a Tyrode solutioncontaining 10 //.g/ml DPH and 5.6 mM K + .However, it was always possible to record arelatively stable Er of approximately 75 to 77mv (Table 1) under these conditions.

Action Potential Overshoot

The effect of DPH (1, 10 /ig/ml) on actionpotential overshoot in altered K+ solutions is

TABLE 1

Potassium-Dependent Effect of DPH (10 ng/ml) on Membrane Resting Potential (mv)

K + (mM)

2.6

3.6

4.6

5.6

DlPHCsr/ml)

10

10

10

10

0.2/sec

89.7 ± 0.789.4 ± 1.1

87.4 ± 1.087.6 ± 1.5

84.2 ±1.984.5 ± 2.0

78.2 ± 2.177.1 ± 1.5

Stimulation1/sec

89.6 ± 1.089.5 ± 1.6

. 88.2 ± 1.387.1 ± 1.0

84.2 ± 1.384.3 ± 1.6

78.3 ± 3.177.4 ± 2.00

frequency2/sec

89.4 ±88.9 ±

87.8 ±86.9 ±

84.7 ±83.3 ±

77.4 ±76.2 ±

0.80.9

1.12.0

1.12.3

3.22.4

3/sec

88.9 ±1 .088.2 ± 2.0

87.9 ± 1.586.4 ±1.2

83.9 ± 2.183.1 ± 2.0

76.9 ± 2.476.5 ± 3.3

Mean values ± SE recorded from 5 preparations (minimum of 13 and maximum of 38 observa-tions at each point).



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20 JENSEN, KATZUNG

9 9 ' - t, l_JJg/ml DPB' | j

p H ?— '^lOug/miDPH !

0.2 1 2Stimulation 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) overshootat 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 recordedfrom 5 rabbit left atrial preparations (minimum of 18and maximum of 23 observations at each point).Tyrode's solution; 36° C ± 0.5°C.

graphically illustrated in Figure 1. Meanvalues ± SE are shown for a minimum of 18and a maximum of 23 observations at eachpoint. Under drug-free conditions, increasingextracellular [K+] produced a progressivedecrease in the magnitude of overshoot. Thelowest concentration of DPH (1 /Ltg/ml) hadlittle effect on K+-dependent changes inovershoot except at 4.6 and 5.6 mM K + , wheresometimes it reversed the depression thatresulted in the increased [K+]. The highestconcentration of DPH (10 jug/ml) had littleeffect on overshoot in 2.6 mM K+ Tyrode'ssolution (Fig. 1), but substantially decreasedit when extracellular [K+] was raised to 4.6 or5.6 mM (particularly at higher driving rates).

Action Potential Rise Time

Following Weidmann's initial study (23)using Purkinje fibers, it has been shown invarious cardiac tissue (24) that, within certainlimits, the maximum rate of rise of phase 0 ofthe action potential is related to the mem-brane potential from which the action poten-tial arises. In the present investigation withrabbit atria, the effects of DPH on phase 0 ofthe action potential (both quantitative andqualitative) depended greatly on extracellular[K+] (primarily, it appears, through changesin Er), drug concentration, and stimulationrate. Representative records are illustrated inFigures 2 and 3 (high sweep speed records),which show superimposed tracings of actionpotentials recorded, in each case, from a singlefiber in various extracellular K+ solutionsbefore and during perfusion with DPH. InFigure 2 the rate of rise of the action potentialis increased over the drug-free value by 1jiig/ml DPH at stimulation frequencies of 2and 3/sec in 5.6 mM K+ Tyrode's solution. Bycontrast the same concentration of DPHexerted no visible effect on the actionpotential upstroke in 2.6 mM Tyrode's solu-tion, regardless of frequency. In the experi-ment illustrated in Figure 3, 10 jug/ml DPHexerted an obvious depressant effect onmembrane depolarization in both 3.6 and 4.6mM K+ Tyrode's solution. It C ± 0.5° C .this effect varies substantially with both theextracellular [K + ] and stimulation rate.

When viewed at high sweep speeds, theupstroke of the action potential consistsroughly of three segments of voltage changeswith time: an initial slow foot, a rapid, almostlinear phase, and a slowly curving terminalphase. Measurements of action potential risetime of 10 to 50% (RT 10-50), and 50 to 90%(RT 50-90) depolarization provided a meansof determining if DPH exerted differentialeffects on slow voltage changes at the foot andthe peak of the action potential. Potassiumand frequency-dependent effects of DPH onRT 10-50 and RT 50-90 recorded from 24atrial preparations are summarized in Figure4. The lowest concentration of DPH (1/xg/ml) has little effect on either segment in



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2.6 mM K' - 1 ug/ml DPH

0.2/sec 2/sec 2/sec 3/sec

5.6 mM K - I jjg/ml DPHyg/rr

0.2/sec l/sec 3/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 stimulationfrequencies (0.2-3/sec). Rabbit left atrium. 36°C. Superimposed tracings of action potentialsrecorded 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'ssolution (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 mMK+, particularly the latter. The effects of 10/xg/ml, and in many cases that of 5 fig/ml, aregenerally similar to those expected withquinidine under comparable conditions. In 4.6and 5.6 mM K+ Tyrode's solution it isparticularly evident (Fig. 4) that both RT 10-50 and RT 50-90 are prolonged by 10 fig/mlDPH. Changes in RT 10-50 appear to beslightly greater than those in RT 50-90.

Action Potential Duration

Representative records of potassium andfrequency-dependent effects of DPH on therepolarization phase of action potentials re-corded from two atrial fibers are shown in theslow sweep speed tracings in Figures 2 and 3.Similar effects of DPH on action potentialduration measured at 50% repolarizationare quantitatively summarized in Table 2.Changes in duration of the action potential


were determined in both control and testsolutions in 26 preparations. In the experi-ments presented in Figures 2 and 3, thisduration is either unchanged or shortened inthe presence of DPH, depending (as didaction potential overshoot and rise time) ondrug concentration, extracellular [K+], andstimulation frequency. In the range of concen-trations studied, both DPH and increased[K+] (separately or in combination) usuallyshortened duration of the action potential atstimulation rates of 1-3/sec. In addition, themagnitude of shortening produced by onedepended critically on the concentration ofthe other. In the presence of 2.6 mM K + , DPHalways shortened duration of the actionpotential with respect to drug-free values,except at the lowest stimulation frequency(Table 2). By contrast, the drug had little orno effect on duration of the action potential in5.6 mM K+ Tyrode's solution, when the actionpotential duration was diminished by aug-


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22 JENSEN, KATZUNG

3.6 mM K + - 10 Mfl/ml DPH

of,

0.2/sec 2/sec 3/s.

4.6 mM K+- lOug/ml DPH

0.2/sec i/sec 2/sec 3/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 stimulationfrequencies (0.2-3/sec). Rabbit left atrium. 36°C. Superimposed tracings of action potentialsrecorded 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'ssolution (bottom). DF = drug free.

TABLE 2

Potassium-Dependent Effect of DPH on Action Potential Duration (msec)

K +(mM)

2.6

3.6

4.6

5.6

DFH(»»g/ml)

15

10

15

10

15

10

15

10

0.2/sec

7.5 ± 1.07.5 ± 0.47.0 ± 0.37.1 ± 0.7

8.2 ± 0.18.0 ± 0.27.9 ± 0.38.2 ± 0.4

7.5 ± 0.17.9 ± 0.67.0 ± 0.36.8 ± 0.7

6.2 ± 0.36.0 ± 0.25.9 ± 0.36.1 ± 0.7

Stimulation frequency

25.120.618.018.4

22.510.618.018.7

18.117.717.517.2

16.315.716.417.0

l/sec

±0.1±0.7± 1.4± 1.4

± 1.6± 1.3± 1.3± 2.4

± 1.2± 1.3± 1.5± 1.7

±0.6± 1.6± 1.6± 1.4

l/sec33.7 ± 1.528.5 ± 1.528.4 ±3.128.1 ± 2.1

29.9 ± 1.627.4 ± 1.928.0 ± 1.128.6 ± 2.0

26.3 ± 1.526.2 ± 1.625.8 ± 1.324.8 ± 1.7

25.4 ± 1.225.7 ± 1.226.1 ± 1.626.1 ± 2.8

2/sec

33.9 ± 1.526.4 ± 0.926.0 ± 3.726.7 =>= 2.9

29.3 ± 1.527.2 ± 1.326.7 ± 0.627.0 ± 2.0

27.6 ± 1.526.0 ± 1.326.5 ± 0.127.5 ± 1.6

26.6 ± 1.525.9 ± 2.425.5 ± 2.227.8 ± 3.4

Mean values ± SB recorded from 26 preparations (minimum of 19 and maximum of 31 observa-tions at each point).



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8

6

LU

5a.

FIGURE 4

Potassium-dependent effect of DPH on action potential rise time between 10 and 50% (RT10-50) and 50 and 90% (RT 50-90) depolarization at stimulation frequencies of 0.2/sec (topleft), 1/sec (top right), 2/sec (bottom left), and 3/sec (bottom right). Means ± SE compiledfrom experiments on 24 rabbit left atrial preparations (minimum of 31 and maximum of 69observations at each point). *P > 0.05 (both RT 10-50 and RT 50-90); i P > 0.05 (RT 50-90 butnot RT 10-50). Each bar has two components: 10-50% and 50-90% rise time.

mented potassium. In some fibers, duration ofaction potential was slightly prolonged byDPH rather than shortened in 5.6 HIM K +

Tyrode's solution. These effects did not appearto be significant.

In several preliminary experiments wefound that when DPH is administered in thecommercial diluent supplied for parenteral use(propylene glycol, 40%; ethanol, 10%, inwater) duration of the action potential issubstantially increased rather than decreasedin the highest K+ solutions, and only slightlyincreased or unchanged in the lowest K+

solutions. Bigger et al. (6) reported that the


commercial diluent diminished DPH-inducedshortening of the action potential duration incanine Purkinje fibers. These and other results(10) leave little doubt that the diluent per seexerts pharmacologic effects, which, at least inisolated tissue studies, may obscure true DPHresponse.

SODIUM REVERSAL OF THE EFFECT OF DPHON TRANSMEMBRANE ACTION POTENTIALS

It is known that increasing the extracellularsodium concentration will diminish or reversesome of the depressant effects of quinidine onelectrical properties of cardiac tissue (16, 17).In five experiments we investigated the


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24 JENSEN, KATZUNG

5.6 mM K + - 10pg/ml DPH

0.2/sec 1/sec

50

mV

2/sec 3/secFIGURE 5

Reversal of effect of 10 ng/ ml (4 X 1O~5 M) DPH in 5.6 mM K+ Tyrode's solution by increasingextracellular [Na + ] (154-308 min). Superimposed tracings of action potentials recorded fromsingle cell before and during perfusion with increased [Na + ], DPH concentration maintainedconstant. Rabbit left atrium; 36°C; stimulation frequency = 0.2-3/sec.

relationship between extracellular [Na+] andDPH-induced changes in rabbit atrial trans-membrane potentials. Typical results areillustrated in Figure 5. In this experiment a100% increase in extracellular [Na+] (154 to308 mM Na+) significantly antagonized themembrane effects of 10 /ng/ml DPH in 5.6 mMK+ Tyrode's solution. Similar but less markedchanges were produced by increasing extracel-lular [Na+] by 50%. The observed changes inphase 0 and the overshoot of the actionpotential with increased Na+ undoubtedly

account for the sodium reversal of a depres-sant effect of DPH on conduction velocitywhich we reported in a previous study (22).

Discussion

These results show that DPH is capable ofexerting a wide range of effects on transmem-brane electrical properties of isolated rabbitatria, including a quinidine-like depression ofthe depolarization phase of the action poten-tial, an enhancement of the depolarizationphase (i.e., an increase in the rate of



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depolarization), depression or enhancementof the action potential overshoot, and in-creased rate of repolarization of the actionpotential. Both the direction and the magni-tude of DPH effects on these properties werefound to depend primarily upon drug concen-tration. Also, the observed effects were mark-edly sensitive to even small changes inextracellular [ K+ ] or the driving frequency.Finally, we have shown that the depressanteffect of DPH on membrane depolarization,like that of quinidine, may be antagonized byincreasing extracellular [Na+].

A major difficulty encountered in any in-vitro study of drug action is deciding whatconcentrations of the drug in the isolatedtissue chamber correspond to therapeutic andtoxic levels of the drug in man. This questionis particularly important in the interpretationof the present results in view of the substantialvariation in response (qualitative and quanti-tative) with DPH concentration. The doserange used in this study was 1-10 /^g/ml(4 X 10-0-4 X 10-5M) DPH. On the basis ofdata available in the literature (5, 22) it ap-pears that during the period when DPH isexerting its antiarrhythmic effects in animalsand man the plasma concentration of the drugis 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 estab-lished by Zeft et al. (25) that during the firstfew hours after intravenous administration ofa single dose of DPH to pigs, the amount ofthe drug concentrated in myocardial tissue isin reasonable equilibrium with that located inthe blood. In view of these findings we feelthat the concentrations we used (1, 5, and 10/xg/ml bath solution) are at least reasonablyclose to the concentrations achieved in the in-vivo application of DPH.

The results presented in Figures 2 through 4emphasize the importance of DPH concentra-tion as a variable in these studies. In thepresence of the lowest concentration (1/i,g/ml) of DPH and elevated extracellular[K+] (4.6, 5.6 HIM) depolarization rate wasnoticeably increased with respect to drug-freevalues with little or no change in resting


potentials, indicating that membrane respon-siveness was increased under these circum-stances. By contrast, depolarization rate wasalways decreased by the highest concentration(10 /xg/ml) and usually by the intermediateconcentration (5 /xg/ml) under comparableconditions, indicating a decrease in membraneresponsiveness. Bigger and associates (6) andStrauss and co-workers (7) have demon-strated that DPH in a range of 10"8-10-5M

(.0025-2.5 //.g/ml) increases membrane re-sponsiveness in canine Purkinje fibers (6) andrabbit and canine atrial fibers (7), particularlyin preparations that have previously beendepressed by toxic concentrations of thecardiac glycosides, or cooling, or anoxia. Inthe present study with rabbit atria, depressionof membrane depolarization occurred in thepresence of DPH concentrations (2 X 10"5,4 X 10~8M) that, in comparison, might beconsidered excessive for antiarrhythmic re-sponse. However, even if we assume that thisis correct, these results are no less significantfor it is still possible, and indeed pertinent, toconsider that depression of membrane respon-siveness by DPH represents an importanttoxic manifestation of the drug—particularlyin patients with altered plasma K+ levels. Theextracellular [K+] concentrations utilized inthis study varied from 2.6-5.6 mM. BothBigger et al. (6), and Strauss et al. (7) usedsolutions containing 3.0 mM K + , which isbelow the potassium levels at which weusually saw depression of membrane functionby DPH, and somewhat less than the reportedphysiological range of 5.0-5.5 mM (26).

A synergistic relationship between extracel-lular [K+] and the cardiac effects of quinidinehas been documented by a number ofinvestigators. For example, both Holland (27)and Armitage (28) found that the depressanteffects of quinidine on contractile force andspontaneous rate of isolated rabbit atria wereblocked by lowering extracellular [K + ]. Re-cently Watanabe and Dreifus (20) reportedthat prolongation of intra-atrial, A-V nodal,and His-Purkinje conduction time by quini-dine in isolated rabbit atria was antagonized inlow extracellular [K+] and enhanced in high


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26 JENSEN, KATZUNG

extracellular [K+]. Watanabe and associates(19) had previously reported similar results inventricular preparations. Moreover, they cor-related extracellular and transmembrane elec-trical phenomena by showing that depressionof conduction and the maximal rate ofdepolarization by quinidine are simultaneous-ly reversed by lowering extracellular [K+].Our results, both the present ones and thoserecently reported (22), indicate that thedepressant action of DPH on membranefunction is dependent upon extracellular [K+]in a manner which is similar if not identical tothat of quinidine. Indeed it appears that,given the right conditions, a common propertyof DPH and quinidine is depression of therate of depolarization of cardiac cells and thatthis property is antagonized by low K+ andenhanced by high K+ in the surroundingmedium.

Additional evidence indicating that DPHand quinidine may exert depressant effects ondepolarization mechanisms via a commonpathway is presented in Figure 5. In thisexperiment a reversal of the depressant effectof DPH on atrial cell depolarization wasrapidly accomplished by increasing the levelof NaCl in the perfusate in spite of thecontinued presence of DPH in the solution. Asimilar reversal of the depressant effects ofquinidine on rabbit atria by various sodiumsalts (lactate, sulfate, chloride) has beendescribed by Cox and West (16) whoconcluded that reversal resulted from aspecific effect of Na + , rather than the anionsutilized or a change in the osmolarity of thesolution. Examination of their records and ourown shows close similarities. The increase indepolarization rate in elevated [Na+] isgreater than one would expect to result fromincreased resting potential, therefore it wouldbe anticipated that an increase in external[Na+] would exert a favorable effect ondepolarization and conduction, as we havepreviously shown (22), by increasing the Na +

gradient.

We can only speculate on the importance ofthe relationship between DPH and K+, andthat between DPH and Na + , at the present

time. More definite conclusions on the roleplayed by these.ions in therapeutic and toxicresponse to DPH must await detailed electro-physiological studies of K + - and Na +-depen-dent drug effects on refractoriness, automatic-ity, and conduction in various cardiac tissues.We can conclude, however, that the effect ofDPH on atrial transmembrane potentials andconductivity is complex, and depends upon asomewhat delicate balance between drugconcentration, heart rate, and extracellularsodium and potassium.

AcknowledgmentWe wish to thank Miss Margaret J. Ballage for her

interest and assistance in these studies.

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diphenylhydantoin sodium (dilantin sodium)upon ectopic ventricular tachycardia in acutemyocardial infarction. Amer J Physiol 163:505, 1950.

2. SCHERF, D., BLUMENFELD, S., TANER, D., AND

TILDIZ, M.: Effect of diphenylhydantoin sodi-um on atrial flutter and fibrillation provoked byfocal application of aconitine or delphenine.Amer Heart J 60: 937, 1960.

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R. A. JENSEN and B. G. KATZUNGSTIMULATION FREQUENCY, POTASSIUM, AND SODIUM

Electrophysiological Actions of Diphenylhydantoin on Rabbit Atria: DEPENDENCE ON

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