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Electrophysiological effects of cobra cardiotoxin on rabbit heart cells
Toxkar, 1973, Vol . 13, pp . 437 46 . Pet~mon Prea~. Printed in Great Britain.
ELECTROPHYSIOLOGICAL EFFECTS OF CABRA CARDIOTOXIN ON RABBIT HEART CELLS Cxswx-LaNG Ho, C. Y. L~ and H . H . Lu Pharmacological Institute, College of Medicine, National Taiwan University and Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, Republic of China and Department of Biophysics, National Defense Medical Center, Taipei, Taiwan, Republic of China (Accepted jor publication 18 Jure 197 Ct18wx-L~rra Ho, C . Y. LEA and H . H . Lu. Electrophysiological eûects of cobra cardiotoxin on rabbit heart cells. Toxicon 13, 437-446, 1975 .-Effects on transmembrane potentials of exrdiotoxin isolated from Formosan cobra venom wero studied on rabbit atrial cells by means of elexKmphysiological techniques. It was found that cardiotoxin at a concentration of 10'' g per ml caused a progressive and irreversible decrease of the resting potential in myocardial cells starting S min after addition of the toxin . The decrease in maximum diastolic repolarization of the pacemaker type cells occurred much later and to a much less extent . Assaciatod with the docrea.4e of resting potential, the magnitude and rate of rise of the action potential, the overshoot, the limo to 80 ~ repolarization and the spike ionic conductance were also dexx+easod. The effort of the toxin on the membrane potential was not altered by either te:trodotoxin or sodium removal but was inhibited by high calcium. It is suggested that disintegration of the membrane structure is responsible for the membrane depolarization . INTRODUCTION
(Naja naja atra) venom is a highly basic polypeptide, characterized by its high lysine contents . Its molecular weight has been determined to be 6734 on the basis of amino acid analysis (60 residues) (NARITA and Lie, 1970). Cardiotoxin causes contracture of the skeletal muscle in many species and systolic arrest of the isolated frog hearts and rat atria. In the in vivo experiments, cardiotoxin causes various changes in ECG pattern, including prolongation of the P-R interval, decrease in amplitude of the QRS complex and complete A-V block . All the phenomena mentioned above have been considered to be the consequence of an irreversible depolarization of the cell membrane (Lse et al., 1968). Some toxins of animal or plant origin have been reported to depolarize cell membrane by increasing the specific ionic conductance of the membrane (ALBUt2ueRQuE et al., 1971a, b ; PYt~Ax and TuRrr>~e, 1972a, b ; S>:~twhln and NnItnIIesFn, 1973 ; TbRx>~ and FRISIAN, 1974) . The mechanism by which the cell membrane is depolarized by careïiotoxin, however, remains to be elucidated. The present experiment using electrophysiological techniques was undertaken to study how cardiac cells are depolarized by cardiotoxin . CARDIOTOXIN isolated from Formosan cobra
METHODS AND MATERIALS Albino rabbits of either sat, weighing from 1 " 0 to 2"0 kg were killed by a blow on the head. The heart was rapidly removed and bathed with a modified Krebs solution aerated with a mixture of 95 ~ O, -F 5 CO,. Ventricles of the heart were removed . The right and the left atria wen separated along the interatrial septum. The right atrium was prepared so that the sinoatrial nodal region was accessible . The left atrium was immobilized with the endocardial surface upward and driven electrically by square pulses of S msex duration, 30 ~ above the threshold at a ft+equency of 2 per sec. 437 TOYICON1975 Yot. l3
438
CHEWN-LANG HO, C . Y . LEE and H . H . LU
Transmembrane potentials were recorded with conventional glass micrcelectrodes filled with 3 M KCI . The resistances of the micrcelectrodes were between 5 and 20 Ms;2 . The voltage-time tracing was displayed on an oscilloscope (Tektronix 502A) and photographed with a kymographic camera . The velocity of impulse wnduction in the atrium was determined either by measuring the latency of the appearance of the action potential or by measuring the time interval between action potentials recorded from two separated cells. The distance from the stimulating cathode to the recording micrcelectrode or from the first to the second recording microelectrode was measured with an eyepiece grid in the dissecting microscope . For the determination of spike ionic conductance, the voltage-time tracing was displayed in cogjunction with the phase-plane trajectory as described by Jet~rrtucx (1963) . Briefly, the action potential was displayed via the horizontal amplifier of the oscilloscope and at the same time led to an electronic differerentiater . The differentiated signal was then fed into the channel A vertical amplifier. A time base sawtooth voltage derived from another oscilloscope was applied to the channel B vertical amplifier. The dual beam oscilloscope consequently permitted the display of the action potential in the phase-plane on channel A and in the conventional voltage-time tracing on channel B . The bathing solution which had a composition (mM) of NaCI, 1360 ; KCI, 2~8 ; CaCI,, 2~5 ; MgSO,, 1~2 ; KH,PO,, 1'2 ; NaHCO,, 125 ; and dextrose, 5~6 was maintained at 37 f 0~5°C and aerated with a mature of 95 ~ O, and 5 ~ CO, . When the Ca'+ concentration of the solution was raised, the amount of NaHCO, was halved and KH,PO, omitted to avoid precipitation . Na+ free solution was prepared by substituting NaCI with sucrose on an isosmolar basis and omitting NaHC0 8 and KH,PO,. The pH of the solution was adjusted to 7~4 with 5 N HCl after 4 mM Tris(hydroxymethyl)aminomethane had been added (ALauQuEtcQue and Wnxrncx, 1972) . Cardiotoxin (eTx) was isolated from Formosan cobra venom by gradient chromatography on a CMSephadex C-SO column as described previously (LeE et at., 1968) . Traces of phospholipase A were removed by repeated rechromatography on a CM-cellulose column using gradients of ammonium acetate buffer (015-0~9 M) . Tetrodotoxin (7TX) was purchased from Sankyo Co ., Japan . RESULTS
Effects of CTX on the resting and action potentials ofatrial cells
Both resting and action potentials were markedly affected by CTX . A concentration of 10 - a g CTX per ml reduced progressively the resting potential starting at about 5 min after its addition . Associated with the change of the resting potential, the amplitude, overshoot and duration of the action potential were also changed as shown in Figs . 1 and 2. The time to 20~ repolarization (20 ~ APD) was not significantly affected within 60 min of observation . The time to 80 ~ repolarization (80 ~ APD) began to be shortened at about 5 min and the peak effect was reached at 20 min after the addition of CTX. The maximal reduction in duration was about 20 ~ of the control (6064 ~ 1 ~65 msec) . Addition of Bae+ (0~2 mM as chloride) 20 min prior to CTX did not alter the effect of the toxin to any significant extent (Fig . 2). This result seems to indicate that the effect of CTX on the action potential duration (APD) is not due to a specific enhancement of K+ conductance, since BaE+ has been shown to inhibit K+ conductance specifically (SPERELAKIS et al., 1967 ; HERnssn~raYSR and SPERELAKiS, 1970) .
The effect of CTX on the spike ionic conductance Spike ionic conductance (g) of the atrial cell before and during the action of CTX measured by means of analysis of the phase-plane trajectory (J&NERICK, 1963, 1964) .
was The results are summarized in Table 1 . The progressive decrease in spike ionic conductance was found to be parallel to the decline of the resting potential as was the maximum rate of rise of the upstroke .
Effects of TTX and of Na+ removal on the CTX-induced membrane depolarization
Membrane depolarization induced by CTX could neither be prevented nor be reversed by substituting the Na+ in the bathing solution with sucrose although the rapidity and extent of depolarization were reduced if Na+ was removed before CTX addition (Fig. 3) . Tetrodotoxin in a concentration insufficient to affect atrial action potentials alone greatly roxicoN r9~s vor. r3
Cobra Cardiotoxin and Heart Cells
439
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L`l'X (lU ' g per ml) ADDED AT U TIME ON RES'T'ING MEMHRANB POTENTTAL AMrIaTImE (p---p AND ovTalsxooT (NEGATIVE VALUES DENOTE LEVEL (O-O), H1iLOW ZERO) (~-' -~ OF ATRIAL CELLS AND THE FROTECITVE EFFECTS OF HIGH Ca'+ (TS II1M) ON CTX-1NDUCED INTiIHirION . FIG . 1 EPFECTS OF
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Hollow symbols represent data obtained from cells in normal Ca'+ (2'S mM) solution and full symbols represent data obtained from cells in high Ca'+ (T5 mM) solution . CaCI, was added to bathing solution 20 min before the application of CTX. Each point represents mean f S .E .M . of 2(1-36 observations from 5 animals .
accelerated the depressant action of CTX (Table 2). In the presence of 2 x 10- ' and 2 x 10 -° g TTX per ml complete loss of spike activity was found at 20 min and at 10 min, respectively, after the application of CTX (10-8 g per ml). In the absence of TTX, the same concentration of CTX required more than 60 min to abolish the spike activity (Fig. 4). The time course of membrane depolarization induced by CTX did not seem to be influenced by the presence of TTX (Fig. 5). E,~`ects of Ca ions on the membrane depolarization and spike suppression by CTX Cardiotoxin-induced membrane depolarization and spike suppression were closely related to the Cas+ concentration in the bathing solution. Decreasing the Cas+ concentration accelerated the action of CTX. By contrast, increasing Cae+ concentration retarded it (Fig. 6, also see Fig. 1). The resting potential and spike activity remained almost unchanged for more than 90 min when CaB+ concentration was increased to 7'S mM (Fig. 1). E $ects of CTX on the impulse conduction in the atrium
Since the conduction velocity is directly proportional to the rate of rise of the action potential, a progressive decrease in conduction velocity in the atrium after the application of CTX is predictable. The data obtained from 5 preparations are shown in Fig. 7. It should be emphasized that the values shown in Fig. 7 are only relative ones since impulse propagation among atrial fibers may go such a tortuous way from one point to another that measuring simply the distance between two points might not be accurate in determination of the conduction velocity . T~DXlCON I973 Yol. I!
440
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Time, FICi. 2. EFFECCS OF
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Ba =+ (O'2 mM)
UPON ACTION OF CTX (lO-° g ACTION POTENTIALDURATION (APD).
per ml) ON
ATRIAL CELL
The duration was measured at 20 ~ repolari7ation (20~ APD) and at 80 ~ repolari7ation (80~ APD) of the action potentials . 73aC1, was added to the bathing solution 20 min before the application of CTX. ~-~ : 80 ~ APD in the presence of Ba'+ ; O-O: 80 ~ APD in the absence of Ba'+ ; "-" : 20~ APD in the presence of Ba'+ ; p-p: 20~ APD in the absence of Ba'+ . Each point represents mean f S.E .M . of 30 observations from 3 animals. TABLE 1 . EFFEC]'S OF CARD10iOXIN (CTX) ON PARAbIETERS OF 7IiE PHASE-PLANE TRAJECTORY OF A71tIAL CELL ACRON PO'fENiTAIS
Concentration of CTX was 10_' g per ml. K1 , K, are slopes of the ascending and descending limb of the phase-plane tretjectory. g is the spike ionic conductance . K 8 = C Ki (K, -f_ K~ . C is the membrane capacitance which is assumed to be 2 lif per cm'. V is the maximum rate of rise of the upstroke. Data are presented as mean ~ S.E .M . Figures in parentheses indicate number of tests Time after CtX
Control
-.
X2
6
9eetiag membrane potentie:
4,q:0 .1(36)
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.7 .613 .1(36)
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î" 31C .5 (20)
148.5110 . :"(20)
57 .412 .T (2f)
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"Significantly different from control values (P <, 0'01). E $ects of CTX on the sinoatrial nodal cells
The sinoatrial nodal cells were much more resistant to CfX than were ordinary atrial myocardial cells. At the concentration of 10_ 6 g per ml which normally depressed the atrial TOXICON 1975 Vol. 13
Cobra Cardiotoxin and Heart Cells
44 1
eo
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Time, min (lO- ~ g per ml) ON RESTING MEILOIRANE P01'EN'I'IAL 130TH IN THE PRESENCE AND rN THE AasENCE of Na+. in normal bathing solution ; O-O: in Na+ free solution . Each point represents mean f S.E.M. of 5-15 observations from 3 animals.
FIG. 3 . EFFECTS OF C'TX ~--~ :
fT~ ON SPIKB SIJPPRFS4IIdG ACTION OF CARDIOTOXIN (CTX) x 10 - ' g per ml) was added 20 min prior to CTX (10_s g per ml). K K,, V, g: same indi cations as in Table 1. Data are presented as mean f S.E.M . Figures in parentheses indicate number of tests TAHIB 2 . EFFECTS OF T1:IRODOI'OXIN
TTX (2
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"Significantly different from the control values (P < 0" 05). Note that the suppressing effect of CTX is accelerated as compared to data shown in Table 1,
cell action potential within 10 min, C'TX did not (or only slightly) aûected the nodal cells in 60 min (Fig" 8). Significant changes in maximum diastolic repolarization and total amplitude of the action potential were not observed unti190 min after the addition of the toxin (Table 3). The beating rate expressed as the cycle length of the action potential did not show any significant change within 60 min of C"TX action. A slight decrease in the cycle length was observed in three out of the five preparations tested during later periods (90-120 min). DISCUSSION
From the results of the present study, it seems apparent that the primary action of CTX on atrial cells is to depolarize the membrane . The coincidence is both time course and magnitude between the change in resting potential on one hand and the change in the action potential on the other strongly suggests that all of the changes in action potential, TOXICON 197J Yo1, !3
CHEWN-LANG HO, C. Y. LEE and H. H. LU
442
O-O: CTX 10 -° g per ml ; "-- ~ ~ : CTX 10 -6 g per ml -~ TTX 2 x 10 -' g per ml ; "---" : CTX 10'6 g per ml -~ TTX 2 x 10 -° g per ml . Each point represents mean f S.E .M . of IS observations from 3 animals (TTX treated) or of 2(1-36 observations from S animals (CTX alone) .
E 1 O
.C Q N C O E m
Time, mln TTX ON THB C,TX-INDUCED l1lEMBRANE DEPOLARi~~ TION. O-O: CTX 10 -° g per ml ; /-- ~ ~ : CTX 10 -6 g per ml -I- TTX 2 X 10 - ' g per ml ; " --- " : CTX 10 -° g per ml ~- TTX 2 x 10 -° g per ml . Each point represents mean f S.E .M. of 15 observations from 3 animals (TTX treated) or of 20-36 observations from 5 animals (CTX alone) . FIG. S . THE LACK OF EFFECT OF
e.g . reduction in amplitude and rate of rise of the upstroke and decrease of spike ionic conductance are secondary to the change in resting membrane potential. The dependence of inward current that causes the upstroke upon the level of resting potential is well known TOXICON 1975 Vol. !3
Cobra Cardiotoxin and Heart Cells
443
I OOr
c E m Y ô. a r m ô a 0 0 +. m E
30
10
3
P O
I 0" I
I 03
loq [Cq],,
1
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1
3" 0
FIG. 6. THE RELATIOxsIIm sETwEErI Ca'+ CONCENTRATION IN THE HATj-IING SOLUTION ([Ca],) AND THE TIME REQUDtED FOR C,TX (10_ s g per ml) TO AHOr icw SPIRE PO'IBNTIAL .
The time was measured from addition of CTX to wmplete loss of spike. Each point represents mean f S.E .M . of 5 animals.
eo
E u _r . û c
0 û v ô U
20
0 Time,
min
CTX (LO- ' $ per ml) ON CONDUCTION VEIACITY 1N THE ATRIUM . Each point represents mean f S.E.M. of 2(1-36 observations from 5 animals,
FIG. ~ . EFFECTS OF
(HODGxiN and HLJXLSY, 1952 ; WEmMnxrr, 195 . The hypothesis that explains the importance of the level of resting potential in the genesis of the spike is that the amount of Na+ carriers which can be activated to carry Na+ current during depolarization is determined by the level of the membrane potential ; within limits, the higher the level the more are the 710XICON 1973 Yo1 . l3
44 4
CHEWN-LANG HO, C. Y. LEE and H. H. LU
40 mV 200msee
90
FIa. H. EFFECTS OF CTX ON THE ACTION POTENIIAIS OF SINOAIRIAL CELLS. C is control . Figures under each tracing are time in min after application of CTX 10 -+ g per ml . Different tracings are from different cells in the same preparation . Voltage and time calibrations are shown in the left lower corner. TABLE 3. EFFECTS OF CARDIOTOICIId (CTX) ON SINOATRUL NODAL CELLS
Data are presented as mean f S.E .M. Figures in parentheses indicate number of tests :"ice aft-_ :. .~..~ Wir) ,antr~ : "+a
120
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56 .532 . . . .
6%~ .81? .+~;25)
;81,615,61,25)
53 .6i2 .C ;£°.'.~
58 .933 .4(25)
381,8111 .4(2°,j
'i0 .412 .4(_5~,
59 .113 .5(25)
381.6119 .8(25)
=5 .633 .1~(25)
kg,314,k~(25)
368.819 .6(25)
44 .513 .3 (25)
49 .314 .3 (25)
362,43ll .1(25)
*Significantly different from control values (P < 001).
carriers available for activation . Prolonged depolarization of the membrane would reduce the amount of available carriers by inactivation . There are many factors that are capable of depolarizing membranes. In short, anything that suppresses membrane Na-K pump, decreases membrane K+ permeability, increases membrane permeabilities to cations other than K+ or creates leakage by damaging the membrane would have the effect of depolarizing the membrane. The involvement of a Na-K pump suppression in the depolarization induced by CTX is unlikely because it has recently been found that CTX does not inhibit Na-K activated ATPase obtained from microsomes of rabbit skeletal muscles (Lin Shiau et al., unpublished observation) . Neither is it likely that a specific increase in membrane permeability to a particular ion is involved because addition of Baß+ or TTX, or removal of Na+ fails to alter the general pattern of the membrane potential change . Considering that cobramine B, an `isotoxin' of CTX (LEE et al., 1970) causes a general increase in membrane permeabilities (WOLFF et al., 1968) and that cardiotoxins isolated TOXlCON 1975 Yol, I3
Cobra Cardiotoxin Hand eart Cells
445
from both Naja raja atra and Dendroaspis jamesoni venoms disorganize the internal structure of muscle fibers (Lnl et al., 1972 ; Ducl->Brr et al., 1974), it seems reasonable to assume that the depolarizing effect of CTX is also the result of a general increase in membrane permeabilities to all ions consequent to membrane damage. The fact that increase in Ca'+ concentration in the bathing solution slows down the speed and reduces the magnitude of depolarization induced by CTX agrees with this assumption, because Cas+ is known to be essential both in the maintenance of membrane integrity and in repair of membrane damage (Dél.~z$, 1965 ; MANELtY, 1966 ; D$ Msl.ho, 1973). The irreversible nature of the CTX action also supports this assumption . The enhancement by TTX of the spike suppressing effect of CTX can be explained by the combined action of the selective inhibition of the transient inward current by TTX (N ARe,we,sHI et al., 1964 ; Dun81, et al., 1967) and the inactivation of Na+ carriers resulted from depolarization of the membrane induced by CTX. It has been reported that TTX decrease the resting membrane permeability to Na+ (Fltr~nsnx, 1969, 1971) and antagonizes the depolarizing effect of batrachotoxin which is known to increase membrane permeability to Na+ (AL,suQu>;ItQu$ et a1.,1971b ; Hocnx and A1.auQustQuE, 1971). The lack of effect of TTX on the time course and magnitude of the depolarization caused by CTX indicates that the effect of CTX on the membrane differs from that of batrachotoxin and is more than just to increase Na+ permeability. The finding that substituting Na+ with sucrose in the bathing solution does not prevent but reduces both the rate and magnitude of membrane depolarization induced by CTX is compatible with the assumption that CTX decreases membrane potential by causing a general increase in membrane permeability . Deprivation of the extracellular Na+ would greatly reduce the total chemicoelectric gradients across the membrane and thus decrease the tendency of an inward leakage of the cation which depolarizes membrane. This explanation of CTX action is also consistent with the findings of our previous experiments on skeletal muscle in which we found that CTX caused contracture of the muscle even when Na+ concentration in the bathing solution had been greatly reduced (Llx $HIAU et al., 1973). It has been demonstrated that sinoatrial pacemaker cells are very much different from other heart cells in sensitivity to certain agents . For example, the resistance of a sinoatrial pacemaker cell to TTX (YnMaclst-n and Snxo, 1966) and to K+ (D$ Ma31,l.o and HOFFMAN, 1960) is extremely high as compared to that of an atrial myocardial cell. The diversity in their properties might explain the difference in sensitivity to CTX between these two types of heart cells although the real reason behind the difference is not yet known. According to W»>anxx (1956) and Hor-~ax and Cxnx~.n (1960), the activities of a pacemaker cell can be changed by either one of the three factors, namely the slope of the prepotential, the level of the threshold potential and the level of maximum repolarization . We can not tell exactly how pacemaker activity was accelerated after long period of exposure to CTX (90-120 min). But judging from the fact that acceleration did not occur until the level of maximum diastolic dppolarization decreased (Table 3), it seems safe to state that decrease in maximum diastolic dppolarization level might be, at least in part, responsible for the accelerating effect of CTX. REFERENCFS Ai.HUQuEnQuE, E. X., DALY, J . and Wrrxor, B . (1971a) Hatrachotoxin : chemistry and pharmacology. Science 17Z, 995 . AreuQu~uE, E . X ., Wl~xxicx, J . B . and Saxsoxs, F. M . (1971b) The pharmacology of batrachotoxin. lI. Effect on electrical properties of the mammalian nerve and skeletal muscle membranes . J. PJwrntac. exp. tirer . 176, 511 . TOYICON l97S Yo1 . l 3
44 6
CHEWN-LANG HO, C. Y. LEE and H. H. LU
AtauQurfltQUe, E. X. and Wexxrcrc, J. E. (1972) The pharmacology of batrachotoxin. 1V . Interaction with tetrodotoxin on innervated and chronically denervated rat skeletal muscle. J. Pharmac . exp. Ther . 180, 633. D$ MEr.r .o, W. C. (1973) Membrane sealing in frog skeletal muscle fibers. Proc. natn. Acad. Sci., U.S.A . 70, 982. Ds MErzo, W. C. and HOFPMAN, B. F. (1960) Potassium ions and electrical activity of specialized cardiac fibers . Am . J. Physiol. 199, 1125 . Dkr~ze, J. (1965) Calcium ions and the healing-over of heart fibers. In : Elektrophysiology of the Heart, p. 147, (TACCARDI, B. and M~rtcrrErrt, G., Eds.) . Oxford : Pergamon Press. Ducf>Erv, L. W., Excei.t., B. J., P~TEr., R. and ShaTx, B. (1974) Changes in motor end-plates resulting from muscle fiber necrosis and regeneration : a light and electron microscopic study of the effects of the depolarizing fraction (cardiotoxin) of Dendroaspis jamesoni venom. J. rreurol. Sci. 21, 391. DunEr., J., P~Ex, K., RünEr., R. and TRAUTWEIIV, W. (1967) The effect of tetrodotoxin on the membrane current in cardiac muscle (Purkinje fibers) . Arch. Ges. Physiol. 295, 213. FrtEEMnx, A. R. (1969) Action of tetrodotoxin on the resting potential of the squid giant axon membrane . Fedn Proc. Fedn Am. Socs exp. Biol. 28, 333. Fx~teN, A. R. (1971) Electrophysiological activity of tetrodotoxin on the resting membrane of the squid giant axon . Comp. Blochern . Physiol. 40A, 71 . FttEEeux, S. E. and TurtxEx, R. J. (1972a) A myotoxin secreted by some piscivorous Conus species. Br. J. Pharmac. 46, 329. FYt~nrnx, S. E. and TvxxEtt, R. J. (1972b) Some actions of Conus achatinus toxin on membrane potentials and excitation~ontraction coupling in mammalian muscle. Proc. Aunt. physiol. Soc. 3(2), 210. HEt: vEre, K. and SPERRi . "Yi $, N. (1970) Decrrase in K conductance and depolarization of frog cardiac muscle produced by Ba'+ . Am. J. Physiol. 219, 1108. Honaxnv, A. L. and HUXIEY, A. F. (1952) A quantitative description of membrane current and its application to conduction and excitation in nerve. J. Physiol., Lond. 117, 500. Ho~rxrt, B. F. and Gh~xEr~tn, P. F. (1960) Elektrophysiology of the Heart. New York : McGraw-Hill . Hoa~rt, P. M. and ArsuQuExQUE, E. X. (1971) The pharmacology of batrachotoxin. Iii. Effect on the heart Purkigje fibers . J. Pharmac. exp. Ther. 176, 529. JExExrcx, H. (1963) Phase-plane trajectories of the muscle spike potential. Biophys. J. 3, 363 . JErmucrc, H. (1964) An analysis of the striated muscle fiber action current. Biophys. J. 4, 77. LAr, M. K., Warn, C. Y. and LEE, C. Y. (1972) Local lesions caused by cardiotoxin isolated from Formosan cobra venom. J. Formosan med. Ass. 71, 328. LEE, C. Y., Cxnxa, C. C., Cxnr, T. H., C~rru, P. J. S., Tssxa, T. C. and LEE, S. Y. (1968) Pharmacological properties of cardiotoxin isolated from Formosan cobra venom. Arch . exp . Path. Pharmak. 259, 360. LEE, C. Y., Lua, J. S. and WEr, J. W. (1970) Identification of cardiotoxin with cobramine B, DLF, toxin y and cytotoxin. In : Toxins of Animal and Plant Orlgln, p. 307, (UE VRIES, A. and Kocrrva, E., Eds.). New York : Gordon & Breach. Lirt Sronu, S. Y., Hunxa, M. C. and LEE, C. Y. (1973) A study on cardiotoxin contracture . J. Formosan med. Ass. 72, 558. MAI~tY, J. F. (1966) Effects of Ca ions on membrane . Fedn Proc. Fedn Am . Socs exp. Biol . 25, 1804 . Nxx~r-rnsru, T., MooxE, J. W. and Sarrr, W. (1964) Tetrodotoxin blockage of sodium conductance increase in lobster giant axon . J. gen . Physiol. 47, 965. N~nrrf,, K. and LEE, C. Y. (1970) The amino acid sequence of cardiotoxin from Formosan cobra (Naja raja atra) venom. Blochem. Biophys. Res. Common. 41, 339. $EYAMA, I. and Naxnxasxr, T. (1973) Increase in sodium permeability of squid axon membrane by adrlrydrograyanotoxin . II . J. Pharmac. exp. Ther. 184, 299. SvFanr ercra , N., Scr~tEroErt, M. F. and Henxrs, E. J. (1967) Decreased K conductance produced by Ba'+ in frog sartorius fibers . J. gen . Physiol. 50, 1565 . TURNER, R. J. and FREEMAN, S. E. (1974) Factors affecting the muscle depolarization due to Conus achatinus toxin. Toxicon 12, 49. WEroMANN, S. (1955) Effects of calcium ions and local anesthetics on elech~ical properties of Purkinje fibers. J. Physiol., Lond. 129, 568. WEroMANN, S. (1956) Elektrophysiologie der Herzmuskelfaser . Bern : Huber. Wor.rx, J., SAr.AHt, R., AMiarros, M. and LaxsEN, P. R. (1968) T'he basic proteins of cobra venom. 11 . Mechanism of action of wbramine B on thyroid tissue . J. biol . Chem . 243, 1290. YAMAOrsrrr, S. and SANO, T. (1966) Effect of tetrodotoxin on the pacemaker action potential of the sinus node. Proc. Jap. Acad. 42, 1194 .
TOXICON 1975 Vol. 13
ELECTROPHYSIOLOGICAL EFFECTS OF CABRA CARDIOTOXIN ON RABBIT HEART CELLS Cxswx-LaNG Ho, C. Y. L~ and H . H . Lu Pharmacological Institute, College of Medicine, National Taiwan University and Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, Republic of China and Department of Biophysics, National Defense Medical Center, Taipei, Taiwan, Republic of China (Accepted jor publication 18 Jure 197 Ct18wx-L~rra Ho, C . Y. LEA and H . H . Lu. Electrophysiological eûects of cobra cardiotoxin on rabbit heart cells. Toxicon 13, 437-446, 1975 .-Effects on transmembrane potentials of exrdiotoxin isolated from Formosan cobra venom wero studied on rabbit atrial cells by means of elexKmphysiological techniques. It was found that cardiotoxin at a concentration of 10'' g per ml caused a progressive and irreversible decrease of the resting potential in myocardial cells starting S min after addition of the toxin . The decrease in maximum diastolic repolarization of the pacemaker type cells occurred much later and to a much less extent . Assaciatod with the docrea.4e of resting potential, the magnitude and rate of rise of the action potential, the overshoot, the limo to 80 ~ repolarization and the spike ionic conductance were also dexx+easod. The effort of the toxin on the membrane potential was not altered by either te:trodotoxin or sodium removal but was inhibited by high calcium. It is suggested that disintegration of the membrane structure is responsible for the membrane depolarization . INTRODUCTION
(Naja naja atra) venom is a highly basic polypeptide, characterized by its high lysine contents . Its molecular weight has been determined to be 6734 on the basis of amino acid analysis (60 residues) (NARITA and Lie, 1970). Cardiotoxin causes contracture of the skeletal muscle in many species and systolic arrest of the isolated frog hearts and rat atria. In the in vivo experiments, cardiotoxin causes various changes in ECG pattern, including prolongation of the P-R interval, decrease in amplitude of the QRS complex and complete A-V block . All the phenomena mentioned above have been considered to be the consequence of an irreversible depolarization of the cell membrane (Lse et al., 1968). Some toxins of animal or plant origin have been reported to depolarize cell membrane by increasing the specific ionic conductance of the membrane (ALBUt2ueRQuE et al., 1971a, b ; PYt~Ax and TuRrr>~e, 1972a, b ; S>:~twhln and NnItnIIesFn, 1973 ; TbRx>~ and FRISIAN, 1974) . The mechanism by which the cell membrane is depolarized by careïiotoxin, however, remains to be elucidated. The present experiment using electrophysiological techniques was undertaken to study how cardiac cells are depolarized by cardiotoxin . CARDIOTOXIN isolated from Formosan cobra
METHODS AND MATERIALS Albino rabbits of either sat, weighing from 1 " 0 to 2"0 kg were killed by a blow on the head. The heart was rapidly removed and bathed with a modified Krebs solution aerated with a mixture of 95 ~ O, -F 5 CO,. Ventricles of the heart were removed . The right and the left atria wen separated along the interatrial septum. The right atrium was prepared so that the sinoatrial nodal region was accessible . The left atrium was immobilized with the endocardial surface upward and driven electrically by square pulses of S msex duration, 30 ~ above the threshold at a ft+equency of 2 per sec. 437 TOYICON1975 Yot. l3
438
CHEWN-LANG HO, C . Y . LEE and H . H . LU
Transmembrane potentials were recorded with conventional glass micrcelectrodes filled with 3 M KCI . The resistances of the micrcelectrodes were between 5 and 20 Ms;2 . The voltage-time tracing was displayed on an oscilloscope (Tektronix 502A) and photographed with a kymographic camera . The velocity of impulse wnduction in the atrium was determined either by measuring the latency of the appearance of the action potential or by measuring the time interval between action potentials recorded from two separated cells. The distance from the stimulating cathode to the recording micrcelectrode or from the first to the second recording microelectrode was measured with an eyepiece grid in the dissecting microscope . For the determination of spike ionic conductance, the voltage-time tracing was displayed in cogjunction with the phase-plane trajectory as described by Jet~rrtucx (1963) . Briefly, the action potential was displayed via the horizontal amplifier of the oscilloscope and at the same time led to an electronic differerentiater . The differentiated signal was then fed into the channel A vertical amplifier. A time base sawtooth voltage derived from another oscilloscope was applied to the channel B vertical amplifier. The dual beam oscilloscope consequently permitted the display of the action potential in the phase-plane on channel A and in the conventional voltage-time tracing on channel B . The bathing solution which had a composition (mM) of NaCI, 1360 ; KCI, 2~8 ; CaCI,, 2~5 ; MgSO,, 1~2 ; KH,PO,, 1'2 ; NaHCO,, 125 ; and dextrose, 5~6 was maintained at 37 f 0~5°C and aerated with a mature of 95 ~ O, and 5 ~ CO, . When the Ca'+ concentration of the solution was raised, the amount of NaHCO, was halved and KH,PO, omitted to avoid precipitation . Na+ free solution was prepared by substituting NaCI with sucrose on an isosmolar basis and omitting NaHC0 8 and KH,PO,. The pH of the solution was adjusted to 7~4 with 5 N HCl after 4 mM Tris(hydroxymethyl)aminomethane had been added (ALauQuEtcQue and Wnxrncx, 1972) . Cardiotoxin (eTx) was isolated from Formosan cobra venom by gradient chromatography on a CMSephadex C-SO column as described previously (LeE et at., 1968) . Traces of phospholipase A were removed by repeated rechromatography on a CM-cellulose column using gradients of ammonium acetate buffer (015-0~9 M) . Tetrodotoxin (7TX) was purchased from Sankyo Co ., Japan . RESULTS
Effects of CTX on the resting and action potentials ofatrial cells
Both resting and action potentials were markedly affected by CTX . A concentration of 10 - a g CTX per ml reduced progressively the resting potential starting at about 5 min after its addition . Associated with the change of the resting potential, the amplitude, overshoot and duration of the action potential were also changed as shown in Figs . 1 and 2. The time to 20~ repolarization (20 ~ APD) was not significantly affected within 60 min of observation . The time to 80 ~ repolarization (80 ~ APD) began to be shortened at about 5 min and the peak effect was reached at 20 min after the addition of CTX. The maximal reduction in duration was about 20 ~ of the control (6064 ~ 1 ~65 msec) . Addition of Bae+ (0~2 mM as chloride) 20 min prior to CTX did not alter the effect of the toxin to any significant extent (Fig . 2). This result seems to indicate that the effect of CTX on the action potential duration (APD) is not due to a specific enhancement of K+ conductance, since BaE+ has been shown to inhibit K+ conductance specifically (SPERELAKIS et al., 1967 ; HERnssn~raYSR and SPERELAKiS, 1970) .
The effect of CTX on the spike ionic conductance Spike ionic conductance (g) of the atrial cell before and during the action of CTX measured by means of analysis of the phase-plane trajectory (J&NERICK, 1963, 1964) .
was The results are summarized in Table 1 . The progressive decrease in spike ionic conductance was found to be parallel to the decline of the resting potential as was the maximum rate of rise of the upstroke .
Effects of TTX and of Na+ removal on the CTX-induced membrane depolarization
Membrane depolarization induced by CTX could neither be prevented nor be reversed by substituting the Na+ in the bathing solution with sucrose although the rapidity and extent of depolarization were reduced if Na+ was removed before CTX addition (Fig. 3) . Tetrodotoxin in a concentration insufficient to affect atrial action potentials alone greatly roxicoN r9~s vor. r3
Cobra Cardiotoxin and Heart Cells
439
20 E
10> B
ô `c ôa
0 0 0 L
d C O
N
â Ed E
0
c0 F
-
L`l'X (lU ' g per ml) ADDED AT U TIME ON RES'T'ING MEMHRANB POTENTTAL AMrIaTImE (p---p AND ovTalsxooT (NEGATIVE VALUES DENOTE LEVEL (O-O), H1iLOW ZERO) (~-' -~ OF ATRIAL CELLS AND THE FROTECITVE EFFECTS OF HIGH Ca'+ (TS II1M) ON CTX-1NDUCED INTiIHirION . FIG . 1 EPFECTS OF
sT>ncE
Hollow symbols represent data obtained from cells in normal Ca'+ (2'S mM) solution and full symbols represent data obtained from cells in high Ca'+ (T5 mM) solution . CaCI, was added to bathing solution 20 min before the application of CTX. Each point represents mean f S .E .M . of 2(1-36 observations from 5 animals .
accelerated the depressant action of CTX (Table 2). In the presence of 2 x 10- ' and 2 x 10 -° g TTX per ml complete loss of spike activity was found at 20 min and at 10 min, respectively, after the application of CTX (10-8 g per ml). In the absence of TTX, the same concentration of CTX required more than 60 min to abolish the spike activity (Fig. 4). The time course of membrane depolarization induced by CTX did not seem to be influenced by the presence of TTX (Fig. 5). E,~`ects of Ca ions on the membrane depolarization and spike suppression by CTX Cardiotoxin-induced membrane depolarization and spike suppression were closely related to the Cas+ concentration in the bathing solution. Decreasing the Cas+ concentration accelerated the action of CTX. By contrast, increasing Cae+ concentration retarded it (Fig. 6, also see Fig. 1). The resting potential and spike activity remained almost unchanged for more than 90 min when CaB+ concentration was increased to 7'S mM (Fig. 1). E $ects of CTX on the impulse conduction in the atrium
Since the conduction velocity is directly proportional to the rate of rise of the action potential, a progressive decrease in conduction velocity in the atrium after the application of CTX is predictable. The data obtained from 5 preparations are shown in Fig. 7. It should be emphasized that the values shown in Fig. 7 are only relative ones since impulse propagation among atrial fibers may go such a tortuous way from one point to another that measuring simply the distance between two points might not be accurate in determination of the conduction velocity . T~DXlCON I973 Yol. I!
440
CHEWN-LANG HO, C. Y. LEE and H. H. LU eo
sa ar E 0
ao
a a zo
Time, FICi. 2. EFFECCS OF
sd
~~b
0
min
Ba =+ (O'2 mM)
UPON ACTION OF CTX (lO-° g ACTION POTENTIALDURATION (APD).
per ml) ON
ATRIAL CELL
The duration was measured at 20 ~ repolari7ation (20~ APD) and at 80 ~ repolari7ation (80~ APD) of the action potentials . 73aC1, was added to the bathing solution 20 min before the application of CTX. ~-~ : 80 ~ APD in the presence of Ba'+ ; O-O: 80 ~ APD in the absence of Ba'+ ; "-" : 20~ APD in the presence of Ba'+ ; p-p: 20~ APD in the absence of Ba'+ . Each point represents mean f S.E .M . of 30 observations from 3 animals. TABLE 1 . EFFEC]'S OF CARD10iOXIN (CTX) ON PARAbIETERS OF 7IiE PHASE-PLANE TRAJECTORY OF A71tIAL CELL ACRON PO'fENiTAIS
Concentration of CTX was 10_' g per ml. K1 , K, are slopes of the ascending and descending limb of the phase-plane tretjectory. g is the spike ionic conductance . K 8 = C Ki (K, -f_ K~ . C is the membrane capacitance which is assumed to be 2 lif per cm'. V is the maximum rate of rise of the upstroke. Data are presented as mean ~ S.E .M . Figures in parentheses indicate number of tests Time after CtX
Control
-.
X2
6
9eetiag membrane potentie:
4,q:0 .1(36)
~ .i :o .4.(36)
2o ;.Gx8 .2(36)
.7 .613 .1(36)
'6 .Et:.3(36!
.? .610 .2~(~~" ;
î" 31C .5 (20)
148.5110 . :"(20)
57 .412 .T (2f)
(2~)
_'. . ;x3 .fi(2C1 n ~3 .s"g,', (zo ;
~L .3 "S.~ e ( :~i
13 .cx_,3+(20)
wT .°_x~~ . :e(20)
. .e1. ,ß(2a ;
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3.21o .2e(2o)
n d.s1o . ; (2a)
20
1.510 .2 (20)
. .31C .?~i2o)
:,e
1.310,2e (~o ;
._10 .3 (zC)
_~ . .32.~~(20 ;
ai :
< 0.01 ",~~,
~ . . . _e(2'.~j
. . .!1 , . . ,
lo5.c17i .-
o
~ . .__ ,
a:.z12 . ;~ ;
.
:6 .1tL . ;:~, .
"Significantly different from control values (P <, 0'01). E $ects of CTX on the sinoatrial nodal cells
The sinoatrial nodal cells were much more resistant to CfX than were ordinary atrial myocardial cells. At the concentration of 10_ 6 g per ml which normally depressed the atrial TOXICON 1975 Vol. 13
Cobra Cardiotoxin and Heart Cells
44 1
eo
E
60
ô t c m
n
40
m c 0
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m
20
0
I 0
v
v
I 30
v
I 80
1
.
~
I 90
Time, min (lO- ~ g per ml) ON RESTING MEILOIRANE P01'EN'I'IAL 130TH IN THE PRESENCE AND rN THE AasENCE of Na+. in normal bathing solution ; O-O: in Na+ free solution . Each point represents mean f S.E.M. of 5-15 observations from 3 animals.
FIG. 3 . EFFECTS OF C'TX ~--~ :
fT~ ON SPIKB SIJPPRFS4IIdG ACTION OF CARDIOTOXIN (CTX) x 10 - ' g per ml) was added 20 min prior to CTX (10_s g per ml). K K,, V, g: same indi cations as in Table 1. Data are presented as mean f S.E.M . Figures in parentheses indicate number of tests TAHIB 2 . EFFECTS OF T1:IRODOI'OXIN
TTX (2
"eutlng -I .
(rse^
. : :~tr .l .
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c + r "".! i nir. ~:
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...?=_ . .açl
i
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(a ;
u .'~_.. .'i " .~: .
~ o
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n
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:.'7~, .
laeec
~
n
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. .
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,`~ .~3 ::. . . . . . . ~ ._ . .__4 . . . . .
. .'.a : .'_.~
.
._ .')z§. . .~?
~~i1 .,_: . ., . .
7p]kt ri. ._
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. -~1
.~ .,._ .. . ..',1'. ._ .kzl .~l :~._,
_ . .a :' . .."(_'.i ; . . . .:_ . .L";1~~' o-~ .Qil .~~ n l:. ~ y '7 ", .
..
:_ .~
. . :~t2 .5
o.
~ .
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"Significantly different from the control values (P < 0" 05). Note that the suppressing effect of CTX is accelerated as compared to data shown in Table 1,
cell action potential within 10 min, C'TX did not (or only slightly) aûected the nodal cells in 60 min (Fig" 8). Significant changes in maximum diastolic repolarization and total amplitude of the action potential were not observed unti190 min after the addition of the toxin (Table 3). The beating rate expressed as the cycle length of the action potential did not show any significant change within 60 min of C"TX action. A slight decrease in the cycle length was observed in three out of the five preparations tested during later periods (90-120 min). DISCUSSION
From the results of the present study, it seems apparent that the primary action of CTX on atrial cells is to depolarize the membrane . The coincidence is both time course and magnitude between the change in resting potential on one hand and the change in the action potential on the other strongly suggests that all of the changes in action potential, TOXICON 197J Yo1, !3
CHEWN-LANG HO, C. Y. LEE and H. H. LU
442
O-O: CTX 10 -° g per ml ; "-- ~ ~ : CTX 10 -6 g per ml -~ TTX 2 x 10 -' g per ml ; "---" : CTX 10'6 g per ml -~ TTX 2 x 10 -° g per ml . Each point represents mean f S.E .M . of IS observations from 3 animals (TTX treated) or of 2(1-36 observations from S animals (CTX alone) .
E 1 O
.C Q N C O E m
Time, mln TTX ON THB C,TX-INDUCED l1lEMBRANE DEPOLARi~~ TION. O-O: CTX 10 -° g per ml ; /-- ~ ~ : CTX 10 -6 g per ml -I- TTX 2 X 10 - ' g per ml ; " --- " : CTX 10 -° g per ml ~- TTX 2 x 10 -° g per ml . Each point represents mean f S.E .M. of 15 observations from 3 animals (TTX treated) or of 20-36 observations from 5 animals (CTX alone) . FIG. S . THE LACK OF EFFECT OF
e.g . reduction in amplitude and rate of rise of the upstroke and decrease of spike ionic conductance are secondary to the change in resting membrane potential. The dependence of inward current that causes the upstroke upon the level of resting potential is well known TOXICON 1975 Vol. !3
Cobra Cardiotoxin and Heart Cells
443
I OOr
c E m Y ô. a r m ô a 0 0 +. m E
30
10
3
P O
I 0" I
I 03
loq [Cq],,
1
I "0
mM
1
3" 0
FIG. 6. THE RELATIOxsIIm sETwEErI Ca'+ CONCENTRATION IN THE HATj-IING SOLUTION ([Ca],) AND THE TIME REQUDtED FOR C,TX (10_ s g per ml) TO AHOr icw SPIRE PO'IBNTIAL .
The time was measured from addition of CTX to wmplete loss of spike. Each point represents mean f S.E .M . of 5 animals.
eo
E u _r . û c
0 û v ô U
20
0 Time,
min
CTX (LO- ' $ per ml) ON CONDUCTION VEIACITY 1N THE ATRIUM . Each point represents mean f S.E.M. of 2(1-36 observations from 5 animals,
FIG. ~ . EFFECTS OF
(HODGxiN and HLJXLSY, 1952 ; WEmMnxrr, 195 . The hypothesis that explains the importance of the level of resting potential in the genesis of the spike is that the amount of Na+ carriers which can be activated to carry Na+ current during depolarization is determined by the level of the membrane potential ; within limits, the higher the level the more are the 710XICON 1973 Yo1 . l3
44 4
CHEWN-LANG HO, C. Y. LEE and H. H. LU
40 mV 200msee
90
FIa. H. EFFECTS OF CTX ON THE ACTION POTENIIAIS OF SINOAIRIAL CELLS. C is control . Figures under each tracing are time in min after application of CTX 10 -+ g per ml . Different tracings are from different cells in the same preparation . Voltage and time calibrations are shown in the left lower corner. TABLE 3. EFFECTS OF CARDIOTOICIId (CTX) ON SINOATRUL NODAL CELLS
Data are presented as mean f S.E .M. Figures in parentheses indicate number of tests :"ice aft-_ :. .~..~ Wir) ,antr~ : "+a
120
üx " Ciad ; : .~i : ,. ^-.ta : (_ : ;
alike ou1,11tu9e (xV)
Lyc:e langtè ~meac)
56 .532 . . . .
6%~ .81? .+~;25)
;81,615,61,25)
53 .6i2 .C ;£°.'.~
58 .933 .4(25)
381,8111 .4(2°,j
'i0 .412 .4(_5~,
59 .113 .5(25)
381.6119 .8(25)
=5 .633 .1~(25)
kg,314,k~(25)
368.819 .6(25)
44 .513 .3 (25)
49 .314 .3 (25)
362,43ll .1(25)
*Significantly different from control values (P < 001).
carriers available for activation . Prolonged depolarization of the membrane would reduce the amount of available carriers by inactivation . There are many factors that are capable of depolarizing membranes. In short, anything that suppresses membrane Na-K pump, decreases membrane K+ permeability, increases membrane permeabilities to cations other than K+ or creates leakage by damaging the membrane would have the effect of depolarizing the membrane. The involvement of a Na-K pump suppression in the depolarization induced by CTX is unlikely because it has recently been found that CTX does not inhibit Na-K activated ATPase obtained from microsomes of rabbit skeletal muscles (Lin Shiau et al., unpublished observation) . Neither is it likely that a specific increase in membrane permeability to a particular ion is involved because addition of Baß+ or TTX, or removal of Na+ fails to alter the general pattern of the membrane potential change . Considering that cobramine B, an `isotoxin' of CTX (LEE et al., 1970) causes a general increase in membrane permeabilities (WOLFF et al., 1968) and that cardiotoxins isolated TOXlCON 1975 Yol, I3
Cobra Cardiotoxin Hand eart Cells
445
from both Naja raja atra and Dendroaspis jamesoni venoms disorganize the internal structure of muscle fibers (Lnl et al., 1972 ; Ducl->Brr et al., 1974), it seems reasonable to assume that the depolarizing effect of CTX is also the result of a general increase in membrane permeabilities to all ions consequent to membrane damage. The fact that increase in Ca'+ concentration in the bathing solution slows down the speed and reduces the magnitude of depolarization induced by CTX agrees with this assumption, because Cas+ is known to be essential both in the maintenance of membrane integrity and in repair of membrane damage (Dél.~z$, 1965 ; MANELtY, 1966 ; D$ Msl.ho, 1973). The irreversible nature of the CTX action also supports this assumption . The enhancement by TTX of the spike suppressing effect of CTX can be explained by the combined action of the selective inhibition of the transient inward current by TTX (N ARe,we,sHI et al., 1964 ; Dun81, et al., 1967) and the inactivation of Na+ carriers resulted from depolarization of the membrane induced by CTX. It has been reported that TTX decrease the resting membrane permeability to Na+ (Fltr~nsnx, 1969, 1971) and antagonizes the depolarizing effect of batrachotoxin which is known to increase membrane permeability to Na+ (AL,suQu>;ItQu$ et a1.,1971b ; Hocnx and A1.auQustQuE, 1971). The lack of effect of TTX on the time course and magnitude of the depolarization caused by CTX indicates that the effect of CTX on the membrane differs from that of batrachotoxin and is more than just to increase Na+ permeability. The finding that substituting Na+ with sucrose in the bathing solution does not prevent but reduces both the rate and magnitude of membrane depolarization induced by CTX is compatible with the assumption that CTX decreases membrane potential by causing a general increase in membrane permeability . Deprivation of the extracellular Na+ would greatly reduce the total chemicoelectric gradients across the membrane and thus decrease the tendency of an inward leakage of the cation which depolarizes membrane. This explanation of CTX action is also consistent with the findings of our previous experiments on skeletal muscle in which we found that CTX caused contracture of the muscle even when Na+ concentration in the bathing solution had been greatly reduced (Llx $HIAU et al., 1973). It has been demonstrated that sinoatrial pacemaker cells are very much different from other heart cells in sensitivity to certain agents . For example, the resistance of a sinoatrial pacemaker cell to TTX (YnMaclst-n and Snxo, 1966) and to K+ (D$ Ma31,l.o and HOFFMAN, 1960) is extremely high as compared to that of an atrial myocardial cell. The diversity in their properties might explain the difference in sensitivity to CTX between these two types of heart cells although the real reason behind the difference is not yet known. According to W»>anxx (1956) and Hor-~ax and Cxnx~.n (1960), the activities of a pacemaker cell can be changed by either one of the three factors, namely the slope of the prepotential, the level of the threshold potential and the level of maximum repolarization . We can not tell exactly how pacemaker activity was accelerated after long period of exposure to CTX (90-120 min). But judging from the fact that acceleration did not occur until the level of maximum diastolic dppolarization decreased (Table 3), it seems safe to state that decrease in maximum diastolic dppolarization level might be, at least in part, responsible for the accelerating effect of CTX. REFERENCFS Ai.HUQuEnQuE, E. X., DALY, J . and Wrrxor, B . (1971a) Hatrachotoxin : chemistry and pharmacology. Science 17Z, 995 . AreuQu~uE, E . X ., Wl~xxicx, J . B . and Saxsoxs, F. M . (1971b) The pharmacology of batrachotoxin. lI. Effect on electrical properties of the mammalian nerve and skeletal muscle membranes . J. PJwrntac. exp. tirer . 176, 511 . TOYICON l97S Yo1 . l 3
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CHEWN-LANG HO, C. Y. LEE and H. H. LU
AtauQurfltQUe, E. X. and Wexxrcrc, J. E. (1972) The pharmacology of batrachotoxin. 1V . Interaction with tetrodotoxin on innervated and chronically denervated rat skeletal muscle. J. Pharmac . exp. Ther . 180, 633. D$ MEr.r .o, W. C. (1973) Membrane sealing in frog skeletal muscle fibers. Proc. natn. Acad. Sci., U.S.A . 70, 982. Ds MErzo, W. C. and HOFPMAN, B. F. (1960) Potassium ions and electrical activity of specialized cardiac fibers . Am . J. Physiol. 199, 1125 . Dkr~ze, J. (1965) Calcium ions and the healing-over of heart fibers. In : Elektrophysiology of the Heart, p. 147, (TACCARDI, B. and M~rtcrrErrt, G., Eds.) . Oxford : Pergamon Press. Ducf>Erv, L. W., Excei.t., B. J., P~TEr., R. and ShaTx, B. (1974) Changes in motor end-plates resulting from muscle fiber necrosis and regeneration : a light and electron microscopic study of the effects of the depolarizing fraction (cardiotoxin) of Dendroaspis jamesoni venom. J. rreurol. Sci. 21, 391. DunEr., J., P~Ex, K., RünEr., R. and TRAUTWEIIV, W. (1967) The effect of tetrodotoxin on the membrane current in cardiac muscle (Purkinje fibers) . Arch. Ges. Physiol. 295, 213. FrtEEMnx, A. R. (1969) Action of tetrodotoxin on the resting potential of the squid giant axon membrane . Fedn Proc. Fedn Am. Socs exp. Biol. 28, 333. Fx~teN, A. R. (1971) Electrophysiological activity of tetrodotoxin on the resting membrane of the squid giant axon . Comp. Blochern . Physiol. 40A, 71 . FttEEeux, S. E. and TurtxEx, R. J. (1972a) A myotoxin secreted by some piscivorous Conus species. Br. J. Pharmac. 46, 329. FYt~nrnx, S. E. and TvxxEtt, R. J. (1972b) Some actions of Conus achatinus toxin on membrane potentials and excitation~ontraction coupling in mammalian muscle. Proc. Aunt. physiol. Soc. 3(2), 210. HEt: vEre, K. and SPERRi . "Yi $, N. (1970) Decrrase in K conductance and depolarization of frog cardiac muscle produced by Ba'+ . Am. J. Physiol. 219, 1108. Honaxnv, A. L. and HUXIEY, A. F. (1952) A quantitative description of membrane current and its application to conduction and excitation in nerve. J. Physiol., Lond. 117, 500. Ho~rxrt, B. F. and Gh~xEr~tn, P. F. (1960) Elektrophysiology of the Heart. New York : McGraw-Hill . Hoa~rt, P. M. and ArsuQuExQUE, E. X. (1971) The pharmacology of batrachotoxin. Iii. Effect on the heart Purkigje fibers . J. Pharmac. exp. Ther. 176, 529. JExExrcx, H. (1963) Phase-plane trajectories of the muscle spike potential. Biophys. J. 3, 363 . JErmucrc, H. (1964) An analysis of the striated muscle fiber action current. Biophys. J. 4, 77. LAr, M. K., Warn, C. Y. and LEE, C. Y. (1972) Local lesions caused by cardiotoxin isolated from Formosan cobra venom. J. Formosan med. Ass. 71, 328. LEE, C. Y., Cxnxa, C. C., Cxnr, T. H., C~rru, P. J. S., Tssxa, T. C. and LEE, S. Y. (1968) Pharmacological properties of cardiotoxin isolated from Formosan cobra venom. Arch . exp . Path. Pharmak. 259, 360. LEE, C. Y., Lua, J. S. and WEr, J. W. (1970) Identification of cardiotoxin with cobramine B, DLF, toxin y and cytotoxin. In : Toxins of Animal and Plant Orlgln, p. 307, (UE VRIES, A. and Kocrrva, E., Eds.). New York : Gordon & Breach. Lirt Sronu, S. Y., Hunxa, M. C. and LEE, C. Y. (1973) A study on cardiotoxin contracture . J. Formosan med. Ass. 72, 558. MAI~tY, J. F. (1966) Effects of Ca ions on membrane . Fedn Proc. Fedn Am . Socs exp. Biol . 25, 1804 . Nxx~r-rnsru, T., MooxE, J. W. and Sarrr, W. (1964) Tetrodotoxin blockage of sodium conductance increase in lobster giant axon . J. gen . Physiol. 47, 965. N~nrrf,, K. and LEE, C. Y. (1970) The amino acid sequence of cardiotoxin from Formosan cobra (Naja raja atra) venom. Blochem. Biophys. Res. Common. 41, 339. $EYAMA, I. and Naxnxasxr, T. (1973) Increase in sodium permeability of squid axon membrane by adrlrydrograyanotoxin . II . J. Pharmac. exp. Ther. 184, 299. SvFanr ercra , N., Scr~tEroErt, M. F. and Henxrs, E. J. (1967) Decreased K conductance produced by Ba'+ in frog sartorius fibers . J. gen . Physiol. 50, 1565 . TURNER, R. J. and FREEMAN, S. E. (1974) Factors affecting the muscle depolarization due to Conus achatinus toxin. Toxicon 12, 49. WEroMANN, S. (1955) Effects of calcium ions and local anesthetics on elech~ical properties of Purkinje fibers. J. Physiol., Lond. 129, 568. WEroMANN, S. (1956) Elektrophysiologie der Herzmuskelfaser . Bern : Huber. Wor.rx, J., SAr.AHt, R., AMiarros, M. and LaxsEN, P. R. (1968) T'he basic proteins of cobra venom. 11 . Mechanism of action of wbramine B on thyroid tissue . J. biol . Chem . 243, 1290. YAMAOrsrrr, S. and SANO, T. (1966) Effect of tetrodotoxin on the pacemaker action potential of the sinus node. Proc. Jap. Acad. 42, 1194 .
TOXICON 1975 Vol. 13