529

THE JOURNAL OF PHARMACOLOCY AND EXPERIMENTAL THERAPEUTICS

Copyright 0 i97i by The Williams & Wilkins Co.

Vol. 176, No. 3Piintedin U.S.A.

THE PHARMACOLOGY OF BATRACHOTOXIN. III. EFFECT ONTHE HEART PURKINJE FIBERS’

P. M. HOGAN’ AND E. X. ALBUQUERQUE

Deportment of Pharmacology, Schools of Medicine and Dentistry, State lnzversity

of New York at Buffalo, Buffalo, New York

Accepted for publication November 12, 1970

ABSTRACT

HOGAN. P. M. AND E. X. ALBUQUERQUE: The pharmacology of batrachiotoxin. III. Effecton the heart Purkinje fibers. J. Pharmacol. Exp. Ther. 176: 529-537, 1971. The effects

of batrachotoxin (BTX) on the electrogenic properties of Purkinjc fibers isolated fromthe dog heart were studied with intracellular techniques. BTX caused a depolarization of

the Purkinje fiber membrane and in a few cases the membrane potential reached valuesof +20 mV. Application of anodal hyperpolarizing current, 1 aiM Na-sucrose solution or

tetrodotoxin to the Purkinje fiber previously depolarized by BTX, restored the mcmii-

brane potential to values similar to control conditions. High extracellu!ar calcium con-

centration prolonged the time course of the BTX effect but did not prevent it. Prior tothe onset of membrane depolarization, BTX caused an increase in the duration of the

plateau and negative afterpotential of the action potential. Simultaneous with the de-polarization induced by BTX, a decremise in the upstroke velocity and overshoot of the

spike potential occurred together with an increase in the rate of (lepolarization duringthe diastolic interval. When the membrane was depolarized to values less than -20 mV.excitability was lost. These effects of BTX on heart Purkinje fibers were antagonized bytetrodotoxin. The results raise the possibility of the existence of functionally different

sodium channels. It is concluded that BTX acts on the heart Purkinje fiber to selectively

increase sodium permeability.

Batrachotoxin (BTX) has been reported to

cause a large variety of ventricular arrhythmias

in addition to a complete block of atrioventricu-

lar conduction; the predominant effect was the

activation of multifocal ventricular ectopic beats

interrupted by transient ventricular tachyca rdia

or fibrillation (Kayaalp et a!., 1970). A terminal

ventricular fibrillation leading to death of the

animal occurred in one hour or less after the ia.

injection of BTX. It was suggested that the

mechanism of action of BTX on cardiac tissue

was at least in part correlated with the ability

of the poison to cause membrane depolarization

Received for publication July 30, 1970.‘in part by U.S. Public Health Serv-

ice Grants NB-08233 (E.X.A.), Heart Associationof Western New York, Inc. 9505-4580 and U.S.Public Health Sen-ice, National Heart and LungInstitute HE 12780-01 (P.M.H.).

2 Present address: Department of Physiology,Schools of Medicine and Dentistry, State Univer-sitv of New York at Buffalo. Buffalo. N.Y. 14214.

Send reprint requests to: Dr. E. X. Albuquer-que. Department of Pharmacology. Schools ofMedicine and Dentistry. State University of NewYork at Buffalo, 122 Capen Hall, Buffalo. N.Y.14214.

due to a selective increase in sodium permeability

(Kayaalp et al., 1970: Albuquerque et al., 1971).

The objective of the present work was to

analyze in sonic detail the effects of BTX on the

elect rogenic properties of heart Purkinje fibers

of young a(lult (logs. Based on the assumption

that the effect of BTX on Purkinje fibers was

essentially similar to that observed by Xarahashi

et al. (1970) on the giant axon of the squid

Loligo pealii and by E. X. Albuquerque and M.

Sasa (manuscript in preparation) on thie lobster

and crayfish giant axons, particular attention

was given to the action of the poison on the

resting membrane and action potentials of Pur-

kinje fibers. Our results demonstrate that BTX is

a highily toxic compound. Its toxicity was mani-

fested by a complete membrane depolarization

of the Purkinje fibers; however, prior to the de-

polarization significant alteration of the action

potential generat ing mechanism was observed.

These effects of BTX on Purkinje fibers were

antagonize(l by tetrodotoxin (TTX). The possi-

bihitv of two sodium channels functionally differ-

ent is discussed.

E �1

530 HOGAN AND ALBUQUERQUE Vol. 176

METHODS. Animal and preparation. Hearts wereexcised from young adult dogs anesthetized with

sodium pentobarbital (30 mg/kg) injected iv. andwere placed immediately in oxygenated Tyrode’s

solution. Purkinje fibers were obtained from the

ventricles by removing the papillary muscles withattached false tendons. This preparation wasmounted by means of stainless-steel insect pins to

a paraffin block in the tissue bath, and it was

bathed continuously with Tyrode’s solution equil-ibrated with a gas mixture containing 95% O�-

5% CO2.Recording details. The resting membrane poten-

tial and action potential of the heart Purkinje fiber

were recorded by means of conventional glassmicro-electrode techniques. The micro-electrodes

were filled with 3 M KC1, and they had a resist-ance of 10 to 20 megohms. The potentials were

continuously monitored on a Tektronix (type 502)

oscilloscope; representative potentials were dis-played on a Tektronix (type 565) oscilloscope andphotographed with a Grass (model C4) kymo-graph camera. The resting membrane potentialwas continuously recorded on a polygraph. Rou-tine calibration was done by applying 100 mV rec-

tangular pulses between the indifferent electrode

and recording amplifier.

The maximum velocity of the action potentialupstroke was determined by two methods. The first.method involved the graphic display of the action

potential upstroke recorded at a ral)id oscilloscopesweep from which the velocity of the upstrokecould be determined. The second method used afirst. derivative (R = 1 megohm, C = 100 pico-

______#Fic. 1. Diagrammatic representation of a typi-

cal action potential of the heart Purkinje fiber. Thetechnique used to measure the various membranepotentials is indicated. A, resting membrane poten-tial (maximum diastolic potential); B, amplitudeof action potential; C, amplitude of overshoot; D,time to repolarize to -60 mY; E, amplitude ofoutput signal from the electronic differentiatorwhich is proportional to the rate of rise of the ac-tion potential upstroke (dV/dt). Time and voltagecalibrations are indicated.

farmids) circuit for electrical differentiation of theupstroke of the action potential.

Figure 1 shows the features of the Purkinje fiber

potentials measured in this study. Since Purkinjefibers do not exhibit a steady resting potential, the

resting membrane potential was taken as the maxi-

mum level of polarization reached after each ac-tiomi potential. In order to detect a chiange in the

duration of the action potential the time requiredto repolarize to -60 mY was measured. This memis-

urement was made because -60 mY is the approxi-mate minimal level of transmembrane potentialnecessary to elicit conducted action potentials in

Purkinje fibers (Weidman. 1955a; Hoffman et a!.,

1957).The preparation was stimulated by means of a

Tektronix (type 161) pulse generator which dcliv-

erect a rectangular pulse of 1 msec duration at. aregular rate (95/mm) through a silver bipolarelectrode in contact with the endocardial surface.In some experiments depolarizing and hyperpolar-izing current pulses were introduced through asecond micro-electrode and the distance betweenthe recording and stimulating electrodes was keptat 150 to 200 �.

Solution and drugs. The Tyrode’s solution hadthe following composition (mihimolar concentra-

tion): NaC1, 137; KC1. 2.7; CaCl,, 2.7; MgCl,.

0.5; NaH,P04, 1.8; NaHCO3, 25.0; and dextrose,5.5. The pH of the solution was 7.2 to 7.3. Thetemperature of the solution was 37#{176}C.

One millimolar Na-Tyrode’s solution was pre-

pared by isosmotic replacement of sodium by su-crose. When the calcium chloride concentration

was increased to 162 mM, the NaH,P04 was re-100 duced to 0.5 mM and a pH of 72 was kept through-

.!�. out the experiments.BTX and TTX were applied to the preparation

by adding them in appropriate concentration to

normal Tyrode’s solution. For each experiment I

mY mg of crystalline TTX (Sankyo Co. Ltd., Tokyo,Japan) was dissolved in 10 ml of distilled water.From this further dilution was made in physio-logic solution.

RESULTS. Effects of batrachotoxin on the rest-

ing membrane potential of heart Purkinje fibers.

BTX, at concentrations of 1.0 to 5.0 X 10� g/ml,

causes a complete and irreversible depolarization

in all heart Purkinje fibers investigated. The

time course and degree of depolarization pro-

duced by BTX were found to be dose-dependent

(fig. 2). The decrease in resting membrane po-

tential of the Purkinje fibers at the above men-

tioned concentrations occurred about 2.5 minutes

after the poison had reached the experimental

�-40

cr

-80 -

I �ll

1971 BATRACHOTOXIN ON HEART PURKINJE FIBERS 531

chamber. After 120 minutes of exposure to BTX

the Purkinje fibers were depolarized, and the

resting membrane potential was observed to vary

from -10 to +20 mV.

Effect of batrachotoxin on the action potential

generating mechanism of heart Purkinje fibers.

In order to determine the effect of BTX on the

action potential generating mechanism of Pur-

kinje fibers, the preparations were driven at a

rate of 95 pulses/mm in order to prevent possible

changes in the regenerative activity which usu-

ally occurs when the preparations are left beat-

ing spontaneously. However, it is important to

point out that the same sequence of alterations

produced by BTX on the Purkinje fiber macrn-

brane was observed whether or not the tissue was

being driven. In all Purkinje fibers investigated,

BTX caused alterations of the action potential

at concentrations as low as 1.0 X 10� g/ml. In

seven preparations, BTX (5.0 X 10#{176}g/ml)

caused an initial increase in the duratiomi of the

plateau of repolarization after 1.0 minutes, from

a control value of 222 ± 13 msec (mean ± S.D.)

to 305 ± 52 msec. This was also accompanied by

an increase in the negative afterpotential of the

spike. The initial alteration in the duration of

action potentials produced by the poison was not

correlated with any significant effect on the rest-

ing membrane potential, upstroke of the actiomi

potential, magnitude of the overshoot or rate of

depolarization during the diastolic interval.

Figure 3 illustrates in detail the changes in the

action potential configuration produced by 2.0 x10�� g/ml of BTX. A rapid depolarization step

took place when the negative afterpotential ex-

tended through the entire diastolic period leading

to the appearance of a negative prepotential and,

within one beat, the resting membrane potential

decreased about 35 mY. As depolarization pro-

gressed, the slope and the extent of the depolar-

ization during the diastolic interval were in-creased and eventually converted the fiber to :1

true pacemaker whose rate exceeded that of the

drive rate. The Purkinje fibers continued to

beat spontaneously until the resting membrane

potential decreased to values less than -20 mY

��‘hereupon excitability and spontaneous activity

was lost.

To test the possibility that. the alteration and

subsequent cessation of the self-regenerative ac-

tivity produced by BTX was due to an increase

in sodium permeability which brought about a

0 80 160MINUTES

Fia. 2. Effect of batrachotoxin on the rest ingmembrane potential (RMP) of heart Purkinje fi-bers of the dog. The arrow ( 1.) indicates the timeat which batrachotoxin was added to the prepara-tion bath in the following concentrations (gramsncr milliliter); #{149}-�, 5.0 X l0’; Q-�,3.0 X 10�; 0-0, 2.0 X 10�; � 1.0 X10�. Note the appearance of a positive membranepotential at 110 minutes after exposure to the high-est concentration of the poison. Each line repre-sents the mean of at least thiree heart Purkinje fi-hers.

decrease in resting membrane potential, we ap-

plied the following proce(lure. Measurements of

the action potential were made in the same fiber

subjected to different degrees of polarization

umndei’ control conditions and after depolarization

by the poison. The fibers were driven by an ex-

ternal stimulating electrode located near the re-

cording micro-electrode. Under control condi-

tions the action potentials obtained during ap-

plicat ion of cathodal depolarization were similar

to those observed during the depolarization of

Purkinje fibers produced by BTX. Figure 4 illus-trates thie following sequence of events. Prior to

the addition of BTX to the Purkinje fiber prepa-

ration, a control recording of the regenerative ac-

tivity was obtained with the membrane potential

preset at several levels by application of cathodal

depolarization. With the resting membrane po-

tential set at -90 mY, the action potential rose

steeply and its rate of rise was about 540 V/sec

(fig. 4). A subsequent decrease of the membrane

potential towards zero caused a slowing in therate of rise and amplitude of the spike. For

600

0

300

0

0

#{163}

5”

#{163} 5

#{163}

A.

S�-A #{149}

#{149}5.#{149}o

#{149}#{149}S.#{149}.#{149}

0 -80-40Em(mV)

Fia. 4. Effect of batrachotoxin omu the relation-ship between the upstroke velocity (cl1� It) of theaction potential of heart Purkinje fibers and se-lected levels of membrane potential (Em). Thefibers were polarized to the desired level of mem-brane potential before an(l during exposure to ha-trachotoxin (2.0 X 10� g/ml) by passimig a currentthrough a second intracellular mnicroelectrode. Thesymbols indicate the values obtained before (#{149})and after exposure to batrachotoxin for 2.5 (0);5.0 (A); 7.5 (M; and 10.0 minutes (O�.

FI(;. 3. Effect of hat rachot OXlfl on t he tct iOU P0-

tent ial amid its first derivat we recoulid fi’omu t heheart Purkinje fiber of the (log. The records shownare the control (C) and at tIme I imiies iIi(li(at ed (luir-ing exposure to batrachotoxin (2.0 X 10#{176}g’mnl).All records are from the samn( fiber I he prep:mra-

ion was (triven b�’ an external stimiiuihuis applied at

the rate of 95 pulses’min. Ijmper vertical scale.100 mY ; lower vertical scale, 425 V sec horizon-tal scale. 200 msec.

532 HOGAN AND ALBUQUERQUE Uol. 176

example, a depolarization of 23 mY from the

control membrane potential reduced the rate of

rise of t lie spike to :mhout 270 V, sec wit Ii simul-

taneous disappearance of the overshoot. When

BTX was admitted to the l)at hung fluid amid de-

l)olariza t ion had begun, a nodal hyper�)ola rizationof the fiber restored the inemnbrane potential to

the coiit rot level. This method caused an increase

in the (lepolarizing effect of BTX, as revealed by

the fact that while complete depolarization by

the poison in nonpolarized preparations took

about. 120 minutes, in the present case the mciii-

brane l)otential reached values from -10 to 0

mV in 10 to 15 minutes (fig. 5). Omi all occasions,

however, ��huen the membrane potential was re-

store(l by anodal hvperpolarization, the rate of

rise and the amplitude of the action potential

were similar to control (figs. 4 and 5). It is

interesting to note that. after five mimiuites of cx-

postire to BTX (2.0 x 10#{176}g ml), �vhien a (he-

polarization of only 20 mY had occurred, an in-crease in the upstroke velocity of the spike po-

teiitial could be Oh)served if an anodal hvper-

polarization brought the membrane potential tocontrol levels. On many occasioiis during the

action of BTX i�’hien the membrane potential was

set at -SO mY, the rate of rise of the regenera-

tive activity was increased by 35’� fig. 4).

With ammodal hyperpolarization oiie can show

1971 B.�TRACHOTOXIX OX IIEA RT PURKI XJE FIBERS 533

FIG. 5. Effect of anodal hyperpolarization on the heart Purkinje fiber depolarized by batrachotoxin.Record A is the control action potential; records B, C and 1) are the action potentials obtained duringexposure to batrachotoxin (2.0 X 10_i g/m.l) at 5, 10 and 15 minutes, respectively. The arrows (�indicate the beginning of anodal hyperpolarization. The second segment of records B, C amid D arediscontinuous with the first but taken in close temporal sequence. Vertical scale, 100 mY; horizontal

scale, 150 msec.

that the disappearance of the action potential

induced by BTX was directly correlated with a

decrease in membrane potential. This method

was rather unsuitable when the membrane po-

tential was reduced to about 50% from control;

under this condition damage to the tissue and

electrodes was encountered due to the necessity

of passing large hyperpolarizing currents. In a

few cases even when the membrane potential was

as low as -10 mY, anodal hyperpolarization al-

lowed the appearance of spike potentials quanti-

tatively similar to the control spikes (fig. 5).

Effects of batrachotoxin on ionic mechanisms

involved in action potential generating mecha-

nisms. The depolarization of Purkinje fibers

caused by BTX is compatible with the suggestion

that the resting sodium conductance is increased

(Albuquerque et at., 1971; Warnick et al., 1971),

Several experiments were therefore carried out

in which the external sodium concentration was

altered prior to and after the action of BTX on

the Purkinje fiber membrane.

When the Purkinje fibers were treated with 1

mM Na-sucrose solution prior to the addition

of BTX, a hyperpolarization of 2 to 5 mV of the

fiber membrane was observed; upon washing the

preparation with normal Tyrode’s solution, the

resting membrane potential returned to the con-

trol level, i.e., about -90 rnV. In these experi-

Inents, six preparations were exposed to 2.0 X

10#{176}g/ml of BTX for about 15 mintites, the

membrane was depolarized to values of the order

of -25.7 ± 5.3 mY whereas the control resting

membrane potential was -87.7 ± 5.9 mY. Wash-

ing of these preparations with 1 mM Na-sucrose

solution restored the membrane potential to

-86.6 ± 5.3 mV. When the Purkinje fibers were

exposed to normal Tyrode’s after being treated

with 1 mM Na-sucrose solution, the resting

membrane potential returned in about 20 seconds

to the depolarized state with values of -18.9 ±

3.9 mY (fig. 6). It is important to mention thatprolonged exposure to 1 mM Na-sucrose solution

caused a considerable deterioration of the prep-

aration. Therefore, the exposure to the Na#{176}-

deficient solution was never for more than 10

minutes. This time proved to be sufficiently long

to allow a complete recovery of the resting mem-

brane potential of the Purkinje fibers depolarized

by BTX.

Another way in which one could study the par-

ticipation of sodium ions on the regenerative

activity is by using TTX. This toxin interferes

with the self-regenerative activity by selectively

BTX IlmMNaI N�TyrC

-50

0 20

MINUTES

9

0�

ii

-100

FIG. 6. Effect of low extracellular sodium con-centration on the resting membrane potential ofthe heart Purkinje fiber previously exposed to ba-trachotoxin (BTX). The resting membrane poten-tial (RMP) before exposure to batrachotoxin(2.0 X 10 g/ml) was -85 mV and after 15 min-utes about -25 mY. Normal Tyrode’s solution (N-Tyr).

blocking passive sodium permeability. The con-

centration of TTX that effectively blocks the ac-

tion potential generating mechanisms of the

squid giant axons and lobster stretch receptor

(Narahashi et a!., 1964; Albuquerque and

Grampp, 1968) is rather ineffective on heart

Purkinje fibers (Dudel et a!., 1967). For instance,

1.5 X 10� g/ml of TTX did not block the action

potential of the Purkinje fiber; however, a mem-

brane hyperpolarization of 2 to 5 mY was ob-

served. This concentration of TTX was applied

to the fiber for 15 minutes prior to the addition

of BTX (1.0 X 10� g/ml) to the TTX-Tyrode’s

solution. Within the scope of our experiments,

this concentration of TTX was sufficient to pre-

vent the effects of BTX on the spike activity and

resting membrane potential for as long as 30

minutes. After 20 minutes exposure to BTX and

TTX, the preparation was washed with normal

Tyrode’s solution; in two minutes depolarization

began and after 20 minutes resting membrane

potential had decreased to -15 mV (fig. 7). Con-

centrations of TTX below 1.5 x 106 g/ml pro-

longed the onset and time course of BTX effects,

but did not prevent the effects of the poison on

534 HOGAN AND ALBUQUERQUE Vol. 176

Purkinje fibers. In other experiments, when a

preparation previously depolarized by BTX was

subjected to TTX (1.5 x 10� g/ml), a complete

repolarization of the fiber membrane to control

levels was recorded. In a few cases the repolar-

ized level exceeded the control level. As the rest-

ing membrane potential increased, the upstroke

velocity, amplitude and duration of the action

potential approached those values recorded in

control conditions. However, under such condi-

tions, the terminal phase of the spike potential

was prolonged and a typical negative afterpo-

tential could be observed (fig. 8).

Calcium ions have been shown to affect mem-

brane permeability to other ions and to exert a

membrane “stabilizing” effect on Purkinje fibers

#{149}j (Weidmann, 1955b). The possibility that high

4 0 extracellular calcium might antagonize the de-

polarizing effect of BTX was therefore tested.

In the experiment illustrated by figure 9, Pur-

kinje fibers were pretreated for 30 minutes with

Tyrode’s solution containing 16.2 mM Ca�. Dur-

ing continued exposure of the preparation to 16.2

mM Ca�-Tyrode’s solution, BTX (2.0 X 10�

g/ml) was applied to the Purkinje fibers. The

presence of high external calcium concentrations

in the physiologic solution delayed the onset and

extent of depolarization produced by BTX but

did not prevent the action of the poison. It is

therefore likely that the action of calcium on the

fiber membrane may be the result of a stabiliz-

ing action of the ion rather than any competitive

interaction with BTX.

DIscussIoN. In the heart Purkinje fibers of

the dog BTX at concentrations of 1.0 to 5.0 x10� g/ml caused complete depolarization of the

fiber membrane and rendered it inexcitable. The

depolarization produced by BTX was irreversible

and persisted after removal of the poison from

the bathing fluid by washing the preparation

with normal Tyrode’s solution.

Several factors may be responsible for the de-

polarization of Purkinje fibers produced by

BTX. First, there is the possibility of an in-

crease in sodium permeability (P�j; secondly,

there is the possibility of a decrease in potassium

permeability (PK), an increase in chloride per-

meability (P�1) and an increase in the leakage

permeability (P1), or a combination of all of

these factors. The possibility that a decrease in

PK would occur after treating the Purkinje fiber

with BTX can be ruled out because TTX, which

BATRACHOTOXIN ON HEART PURKINJE FIBERS1971

0�

535

0

-100

0

-50

-100I I I - 1 _l

-5 0 15 30 45 60

TIME - MINUTESFIG. 7. Typical experiments showimig the ability of tet.rodotoxiiu (TTX) to prevent the depolarizing

action of batrachotoxin (BTX) on heart Purkinje fibers of the dog. The effect of batrachotoxin omi theresting membrane potential (RMP) of a preparation bathed in normal Tyrode’s solution is shownby open circles (O-O) amid the first arrow (1) imidicates the addition of 1.0 X 10� g/ml of the poison.The fiber began to depolarize after five mimiutes. In another preparatiomi tetrodotoxin (1.5 X 106 g/ml)was added to the baihimig media at zero time, and the action potentials with their first. derivatives(upper records) amid the resting membrane potential (.-.) were simultaneously recorded. The dotunder each action potential indicates the time at which the record was taken amid corresponds to theabscissa. After 15 minutes, batrachotoxin (1.0 X 10� g/ml) was added to the TTX-Tvrode’s solutionas indicated by the second arrow. Removal of both toxins by washing with miormnal Tyrode’s solutionis indicated by the third arrow, and depolarization progressed after two minutes.

affects the earlier transient sodium conductance

of the heart Purkinje fiber (Dudel et al., 1967;

Carmeliet and Vereecke, 1969) and is known to

have no effect on PK, was able to restore the rest-

ing membrane pot.eiitial completely. Therefore,

if the depolarization produced by BTX was due

to a reduction of PK, tetrodotoxin would have

been ineffective in restoring the membrane po-

tential. Furthermore, if a decrease in PK was

the factor involved in the depolarization of the

Purkinje fiber membrane one would expect a

further decrease in membrane potential when

the preparations were washed with 1 mM Na-

sucrose solution for 10 minutes after BTX treat-

ment. In fact, 1 mM Na-sucrose solution re-

stored the membrane potential to values of about

3 mV more negative than in control conditions.

It seems unlikely that increases in Pm and P�1

play a major role in the effects of BTX, because

the poison depolarizes the fibers to near zero

values and in some occasions to positive values

(fig. 2). Since BTX depolarized the heart Pur-

kinje fiber beyond the equilibrium potential for

the leakage component (Es) and the equilibrium

potential for chloride (Efl), the depolarization

then produced by the poison may not be cor-

related with the increase in P� and Pcm. Although

chloride ions play a minor role in resting fibers,

this ion becomes very important as a carrier of

electrical charge during depolarization; however,

this seems to be due to a decrease in potassium

conductance of the surface membrane upon de-

polarization rather than to an increase in chlo-

ride conductance (Carmehiet, 1961). Thus, we

may conclude that BTX caused depolarization

of the heart Purkinje fiber by a selective effect on

the P5k. In order to reinforce this suggestion,

several other experimental findings were ob-

tained. For instance, as is illustrated in figures

7 and 8, BTX effects were antagonized by TTX

and removal of both TTX and BTX was fol-

lowed by immediate membrane depolarization.

Only application of 1 mI’iI Na-sucrose solutions

or TTX could restore membrane potential.

The present findings raise another important

problem regarding the nature of sodium channels

0-

0.3 sec

#{149}50 -

-100 -

0 15 30 45 60 75TIME -MINUTES

Fm;. 8. Effect. of tetrodotoxin (TTX; 1.5 X 10’ g/ml) on the heart Purkimije fiber of the dog previ-ously depolarized by bat rachotoxin (BTX). Resting membrane potential (RMP) under each actionpotemitial record corresponds to the time on the abscissa at which the records were taken. The dot undereach act iou potential record indicates the time at which the record was taken and corresponds to theabscissa. Normal Tyrode’s solution (N-Tyr).

0

>E

�-4C

-8C

1530 45MINUTES

FIG. 9. Typical alteration of time time courseof batrachotoxin (BTX)-induced depolarizationcaused by increased extracellular calcium concen-tration. The curve connecting the open circles(O-O) shows the depolarization caused by ba-trachotoxin (5.0 X l0’ g/ml) in Tyrode’s solutioncontaining normal cxtracellular calcium concentra-lion (2.7 mM). The curve connecting the closedcircles (#{149}-#{149}) shows the decrease in restingmembrane potential (RMP) induced by batracho-toxin after 30 minutes pretreatment and continu-ous perfusion with Tyrode’s solution (‘ontainingextracellular calcium concentration (16.2 mM).The arrow ( �.) indicates the onset of bat rachotoxinperfusion.

536 HOGAN AND ALBUQUERQUE Vol. 176

>

E

a-

N-Tyr� BTXlix N�Tyr TTX N-Tyr

� - amid also provide further enforcement for the

hypothesis which claims the existance of an

heterogeneous electrogenic membrane (Grund-

fest, 1966; Hille, 1968). Although the present

data suggest that resting sodium channels in the

membrane are opened by BTX, the ability of the

fiber membrane to undergo a sodium conduc-

tance increase upon depolarization is unaltered,

provided t.hat the membrane is hyperpolarized

to the coiitrol level. The similarity of the up-

stroke velocity of the action potential initiated

at identical levels of membrane polarization be-

fore and during BTX treatment also suggests

that the poison does not alter the ability of the

surface membrane to generate an increase in

the inward sodium current during spike activa-tion. Furthermore, TTX did not block the action

potential of the Purkinje fiber in a concentration

which antagonized BTX action. Similarly, TTX

application to a preparation previously depolar-

ized to near zero level by BTX restored the

membrane potential and spike activity. In con-

clusion, the present. results showed that the ef-

fect of BTX on heart Purkinje fibers is due to a

selective increase in PN5 and suggest that theresting sodium channels in the electrogenic mem-

1971 BATRACHOTOXIN ON HEART PURKINJE FIBERS 537

brane may be functionally different from the

sodium channels for membrane excitation.

ACKNOWLEDGMENTS. We gratefully acknowl-

edge the helpful discussions and comments of

Dr. J. Daly and Dr. J. E. Warnick as well as

the technical assistance of Miss Dolores Timmer

and Miss Druscilla Latta. The authors were in-

debted to Dr. J. Daly and Dr. B. Witkop for

their generous supply of batrachotoxin used dur-

ing the course of this investigation.

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