Pharmacology 14: 256-264 (1976)
Different Toxic Effects of Ouabain and 16-Epi-Gitoxin on Purkinje Fibres and Ventricular Muscle Fibres Götz Nowak and Knut-Olaf Haustein Institute of Pharmacology and Toxicology, Medical Academy Erfurt, Erfurt
Key Words. Different actions on myocardium and Purkinje system • 16-Epi-gitoxin • Glycoside actions • Isolated Purkinje fibres • Isolated ventricular muscle fibres • Ouabain • Survival time of electrically driven cardiac fibres Abstract. Action potentials of isolated Purkinje fibres and ventricular muscle fibres of canine hearts treated with ouabain and 16-epi-gitoxin were recorded by microelectrodes. Under the influence of both glycosides, Purkinje system fibres became inexcitable earlier than ventricular muscle fibres. Being exposed to 0.\2S ijM ouabain and 2.5 yM 16-epigitoxin, both Purkinje and ventricular muscle fibres became poisoned in the same time as ventricular muscle fibres exposed to 0.1 tiM ouabain and 1.5 yM 16-epi-gitoxin. At the lower 16-epi-gitoxin concentration Purkinje fibres had 2.5 times the survival time of that exposed to the lower ouabain concentration. Compared to 16-epi-gitoxin the inexcitability of Purkinje fibres after ouabain remained irreversible. The semisynthetic glycoside 16-epigitoxin exerts a weaker effect on the specialized conducting system.
Based on a great number of experimental (Cat tell and Gold, 1941; Farah and Maresh, 1948; Gruhzit and Farah, 1953; Takahashi et at., 1936; White and Salter, 1946) and clinical studies (Batterman et al, 1952; Chavez, 1963; Schwiegk, 1960) it is supposed that there are no significant differences between therapeutic, toxic, and lethal doses of common digitalis preparations. However, in recent years it was shown that essential differences exist among natural glyco sides on the one hand (Ito et al, 1970) and between natural and semisynthetic glycosides on the other (Haustein et al., 1970; Haustein and Hauptmann, 1974). In order to obtain glycosides with a modified effect it seemed promising to utilize semisynthetic modifications of the genin moiety, an approach proven useful in other classes of drugs (glucocorticoids, anabolics and others). Comparative studies on 16-epi-gitoxin (Lindig and Repke, 1967), ouabain and gitoxin revealed that the effect of semisynthetic glycosides on excitable
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Received: September 25,1975.
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tissues is much weaker than that of natural glycosides (Haustein el al, 1970). The investigations of Vassalle et al (1962) revealed that after exposure to oua bain Purkinje system fibres isolated from canine hearts were poisoned faster than ventricular muscle fibres. In the present experiments isolated Purkinje fibres and ventricular muscle fibres were incubated with ouabain and 16-epi-gitoxin to elucidate differences in their effects. The microelectrode technique according to Vassalle et al. (1962) was used for recording intracellular potentials. Starting from equieffective glycoside concentrations determined in the guinea pig heart concentrations are used producing a 30 % ( 0.1 hM ouabain, 1.5 juA/ 16-epi-gitoxin) or 50% (0.125 tiM ouabain, 2.5 ¡iM 16-epi-gitoxin) positive inotropic effect. Both ‘high’ glycoside concentrations led to an arrhyth mia frequency (At) 80 (ouabain) and 20% (16-epi-gitoxin), respectively, whereas after exposure to both ‘low’ glycoside concentrations only in the case of ouabain (50%) arrhythmias were observed. To study whether the toxic glyco side effects are reversible glycoside concentrations were used causing a fast maximum response (03 pM ouabain, 5.0 pM 16-epi-gitoxin). The arrhythmia frequency was 90 (ouabain) and 62 % (16-epi-gitoxin), respectively (Haustein et al, 1970). Materials and Methods Preparation Mongrel dogs of either sex between 1 and 3 years of age were anaesthetized with 25 mg sodium hexobarbital per kilogram body weight. After midsternal ihoracotomy the heart was removed, immediately washed in the nutritive medium, both ventricles opened, false ten dons and either trabeculae earneae or small papillary muscles rapidly removed. The prepara tions were transferred to 10 ml tissue baths and fixed with thin platinum electrodes, used for stimulation as well.
Electrophysiological Technique Hither of the two tissue baths contained one Purkinje or one ventricular preparation of the same canine heart. Stimulation was performed with square pulses < 1 2 msec, 0.2 I V) of an Orion-Vl.V function generator (Orion Electronics, Hungary). Action potentials were recorded by microclectrodcs according to Ling and Gerard (1949), filled with 3 M KCI. Tip diameters were less than 1 Mm and internal resistance was 7 10 Msi. The tissue baths were mounted on cross slides of two microscopes the ocular supports of which contained the cathode follow-up amplifiers (difference amplifier with field effect transistors, input resis tance 10M SI) with directly coupled electrodes. Both amplifiers were connected to a Mingograph 34-recorder (Elema-Schonander, Sweden) and a dual beam oscilloscope. Action potentials were recorded at a paper speed of 100 mm/sec. A three-axes stereomicroscope
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Composition o f the Nutritive Medium Nutritive medium composition was the following (in m/W): Na('l 137.0, KC1 5.5, MgC32 1.05, CaClj 2.7, NaHCOj 11.9, NaH2 PO., 1.78. glucose 5.55. The medium was matched to a pH of 7.2 with 0.1 N HC1, continuously bubbled with carbogen (95 % 0 2 /5 % C 02) and kept at 37 °C.
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fixed above the baths was used to observe the insertion of electrodes into the preparation. Action potentials of both preparations were taken from the third and fourth cell layers (passing the electrode through one cell layer abrupt increase of action potential amounting to 120 130 mV can be observed within it). Tissues were stimulated at a rate of 60/min. Recording Procedure After equilibration of 30 60 min the glycosides were added to the baths. Action potentials were recorded at 15-min intervals and mean values were calculated from 10 to 20 potentials, respectively. Duration and amplitude of action potentials were measured. The time interval between starting the glycoside exposure and the occurrence of inexcitability was referred to as survival time. Glycosides Ouabain according to DAB 6 (VEB Arzneimittelwerk, Dresden) and 16-epi-gitoxin (kindly supplied by the Central Institute of Molecular Biology, Academy of Sciences of GDR, Biomembrane Section, Berlin-Buch) were used. Both glycosides were dissolved in highly purified dimethylformamide, the 1 nuW stock solution was diluted with nutritive medium up to final concentration. The final concentration of dimethylformamide used throughout experiments was without any effect on fibre preparations.
Results
Effects o f 'Low ' Glycoside Concentrations on Ventricular Muscle and Purkinje Fibres (fig. 2, 3, table I) Both glycoside concentrations led to a 60 % decrease in action potential duration of ventricular muscle fibres and a 30 % (ouabain) and 50 % ( 16-epigitoxin) amplitude reduction, respectively. If Purkinje fibres are exposed to the above glycoside concentrations, differences in response result. Under the influ ence of ouabain, Purkinje fibres became inexcitable in about 85 min, as was also observed at higher concentration. Action potential duration dropped and the amplitude decreased to about the same values as with high ouabain concentra tion. In comparison, inexcitability appeared 200 min after starting 16-epi-gitoxin exposure. In this case, action potential duration dropped by about 35 % of
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Effects o f ‘High ’ Glycoside Concentrations on Ventricular Muscle and Purkinje Fibres (fig. 1, table I) Doses of 0.125 ¡M ouabain as well as of 2.5 juA/ 16-epi-gitoxin caused elec trical inexcitability of working muscle fibres within the same time. The duration of action potentials dropped about 60 % of the standard and the amplitude was about 50 % of the initial values. Purkinje fibres became inexcitable much faster than ventricular muscle fibres under both glycosides. The duration of action potentials dropped 30 % only, the amplitude, however, was reduced 50 (16-epigitoxin) and 60 % (ouabain), respectively. Ventricular muscle fibres had a sur vival time 2.1 or 2.3 times higher than Purkinje fibres.
259
Toxic Glycoside Effects on Isolated Cardiac Eibres
M inu te s
Fig. I. Effects of 0.125 nM ouabain () and 2.5 /wW 16-cpi-gitoxin (•) respectively on the amplitude (------ ) and duration (------ ) of action potentials of ventricular muscle fibres (A) or Purkinje fibres (B). The signs represent mean values of the experiments of table I.
Table I. Glycoside poisoning of isolated ventricular and Purkinje fibres of the canine heart Glycoside
Concentration
»M
Survival time (min ± SEM)
------------------ventricular muscle fibres
Ouabain
0.1
16-Epi-gitoxin
0.125 1.5 2.5
321 i 57(4) 186 ± 31(4) 343 ± 62(4) 156 ± 21 (4)
Purkinje fibres 81 89 200 68
? 11(5) i 21 (5) t 49 (5) ± 3(3)
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* The two values marked differ significantly (p < 0.001). Number of experiments indicated in parentheses.
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M.nutes
Fig. 2. Effects of 0.1 tiM ouabain (o) and 1.5 uM 16-epi-gitoxin (•) respectively on the amplitude (------ ) and duration (------ ) of action potentials of ventricular muscle fibres (A) or Purkinje fibres (B). The signs represent the mean values of the experiments of table 1.
Reversibility o f Glycoside Effects ( Wash-Out Experiments) (fig. 4) Exposed to both glycoside concentrations, Purkinje fibres became inexcit able in 2 0 -3 0 min. After inexcitability occurred the preparation was washed three times with glycoside-free nutritive medium in 5 min. The ouabain inexcit ability remained irreversible for a further 60 min (fig. 4). In this time the ampli tude increased to 35 mV, additional wash-out procedures, however, did not eliminate toxic glycoside effects in the following 3 h. Compared to ouabain, Purkinje fibres poisoned with 16-epi-gitoxin gener ated first action potentials already 15 min after wash-out. The average amplitude
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initial values, the amplitude, however, decreased 75 %. Significantly lower ampli tudes were observed than with ouabain. Compared to ventricular muscle fibres, Purkinje fibres exposed to ouabain became poisoned 3.9 times, after 16-epigitoxin 1.7 times faster.
261
Toxic Glycoside Effects on Isolated Cardiac Fibres
01pA7 ouabain Ventricle fib re s
1.5
P urkinje fib re s
Ventricle fib re s
16-epi-gitoxm Purkinje fib re s
mm
J\_j Y l \ J \ IY J Y J V J \ !Y_A_ k _ _ K j\ J Y
------------
__ I?
o
J \ J \ , VJ v
i J \_ fY K J N 60
J \_ J V r v A
90
A __rv N _ K
120
A J V .tN _iN
ls e c 150
JV_A_
t\__ tv
180
J\__JV_
A_______ V,
2 *0
A _____ A .
r
/
300 0
JY JY
1 iY _ iV K _ K
Fig. 3. Effects of ouabain and 16-epi-gitoxin on action potentials of electrically driven ventricular muscle and Purkinje fibres of canine hearts in dependence on the time of glyco side incubation. Control: without glycoside incubation electrically driven fibre over the same time as within the incubation experiment.
increased to 93 mV and the duration to 450 msec 60 min after the initiation of this period. Thus, the initial values were nearly regained.
Our studies confirm the results of Vassalle et al. (1962) which revealed that Purkinje fibres of canine hearts became poisoned faster than ventricular fibres. If glycoside concentrations are causing a relatively high increase in contractile force
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Discussion
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(0.125/jA/ ouabain or 2.5 ju/W 16-epi-gitoxin) in the isolated guinea-pig heart, both glycoside concentrations showed no differences in toxic effects on canine hearts. Low concentrations of both glycosides (0.1 nM ouabain and 1.5 /jM 16epi-gitoxin) produced an equieffective moderate inotropic effect in the guineapig heart as well as the same time course of poisoning ventricular muscle fibres (table I). Considering both glycosides, marked differences in this time course were observed in Purkinje fibres. It could be shown that Purkinje fibres exposed to low glycoside concentrations had a survival time 2.5 times higher after 16epi-gitoxin than after ouabain (table 1; p < 0.001). Thus, the hypothesis stated in preceding studies is confirmed that the wider ‘therapeutic range' of 16-epi-
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Fig. 4. The influence of wash-out on poisoning Puikinje fibres with ouabain and 16epi-gitoxin. The amplitude and duration of action potentials restitute only after 16-epigitoxin. The spike potentials are stimulus artefacts as well as the reversed polarity occurring before action potential.
Toxic Glycoside Effects on Isolated Cardiac Fibres
263
gitoxin is based on the weaker effect on the specialized conducting system (Haustein et al., 1970). The temporal differences in the glycoside effect occurrence could be explained by the results obtained in studying the kinetic parameters of the enzyme system (Repke et al., 1973). Accordingly, the delayed onset of Purkinje fibre inexcitability after 16-epi-gitoxin could be due to a much higher dissociation rate of the glycoside-receptor complex. In the (Na* + K+)-ATPase system of the guinea-pig heart, binding constants for the formation of glycosidereceptor complexes only slightly vary whereas the decay of the complex is 30 times higher in the case of 16-epi-gitoxin than in the case of ouabain (Repke etal., 1973). It is supposed that these results obtained with the receptor enzyme of the guinea-pig heart could be applied to the receptor of the Purkinje system. It is concluded that the glycoside-binding receptor areas of the (Na+ + K*)-ATPase of Purkinje and ventricular muscle fibres vary in conformation and possibly in structure. Differences in kinetics between the appearance of the effect and its disappearance or between complex formation and decay could be due to differ ent glycoside-binding receptors. Under the experimental conditions comparable to ours ,Pastelin and Mendez (1972) observed that poisoning of Purkinje fibres with 3|3-Z)-glucopyranosyl14/3-dihydroxy-2 l,23-bis-nor-5/3-chol-20(22)-ene-20-carboxylic acid 7 -lactone (AY 22241), a semisynthetic glycoside, was eliminated just as rapidly as in our studies on 16-epi-gitoxin. The results are not considered reliable because compar ative studies on ventricular muscle fibres or with other glycosides have not been performed yet. If the results obtained with isolated Purkinje and ventricular muscle fibres can be applied to the heart in situ, the following conclusions can be drawn to characterize differences in the effects of ouabain and 16-epi-gitoxin on the ventricular muscle fibre and Purkinje system: provided that the blood level of both glycosides decreases comparatively fast, the conducting system is more susceptible to the effects of ouabain than 16-epi-gitoxin. Action of the latter, however, is mainly directed to the working muscle. The presented results with 16-epi-gitoxin seem to support the hypothesis of Repke et al. (1970) that a wider range of inotropically effective glycoside concentrations following a moderate inhibition percentage of the ATPase system may be reached by semi synthetic modification of the genin moiety (Repke, 1970: Repke et al., 1973). Moreover, a dissociation of glycoside effects on working muscle and specialized conducting system is attained (Haustein et al., 1970).
The authors wish to thank Prof. /. Zell, Institute of Physiology of the Karl-MarxUnivcrsity Leipzig, for introduction to the electrophysiological technique, and Dipl.-lng. IP. Koth for the engineering of the amplifier equipment.
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A cknowledgements
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264
References
Dr. Knut-Olaf Haustein, Institut für Pharmakologie und Toxikologie, Medizinische Akade mie Erfurt, Nordhäuserstrasse 74, DDR 50 Erfurt (GDR)
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Batterman, R.C.; DeGraff A.C., and Rose, O.H.: Therapeutic range of gitalin (amorphous) compared with other digitalis preparations. Circulation 5: 201-207 (1952). Cattcll, M. and Gold, H.: Studies on purified digitalis glycosides. 111. The relationship between therapeutic and toxic potency. J. Pharmac. exp. Ther. 71: 114 125 (1941). Chavez, /.: Comparative value of digitalis and ouabain in treatment of heart failure. Archs intern Med. 72: 168 176 (194 3). Farah, A. and Maresh, G.: Determination of the therapeutic, irregularity, and lethal doses of cardiac glycosides in the heart-lung preparation of the dog. J. Pharmac. exp. Ther. 92: 32-42(1948). Gruhzil, C.C. and Farah, A.E.: Determination of the therapeutic range of gitalin in the heart-lung preparation of the dog. J. Pharmac. exp. Ther. 108: 112 116 (1953). Haustein, K.-O. and Hauptmann, J.: Studies on cardioactive steroids. 11. Structure-activity relationships in the isolated guinea-pig heart. Pharmacology 11: 129 138 (1974). Haustein, K.-O.: Markwardt, and Repke, K.R.H.: Different relationship between thera peutic and toxic actions of 16-epi-gitoxin, gitoxin and ouabain on isolated cardiac preparations. Eur. J. Pharmacol. 10: 1 1 0 (1970). Ito, M„ Hollander, P.B.: Marks, B.H., and Dutta, S.: The effects of six cardiac glycosides on the transmembrane potential and contractile characteristics of the right ventricle of guinea pigs. J. Pharmac. exp. Ther. 172: 188 195 (1970). Undig, C. und Repke, K.: Verfahren zur Herstellung von 16a-Hydroxysteroiden der Cardenolidreihe. DDR-Patentschrift, Wirtschaftspatent, Amtl. Aktenzeichen Wp 12o/128 735 (1967). Ling, G. and Gerard, L.W.: Microelectrodes. J. cell. comp. Physiol. 34: 397 405 (1949). Pastelin, G. and Mendez. R.: Singular effects of a new short acting cardiac glycoside in Purkinje cells. Eur. J. Pharmacol. 19: 291 -293 (1972). Repke, K.R.H.: Biochemische Aspekte der Digitalistherapie. Ber. ges. inn. Med. 7: 8-17 (1970). Repke, K.R.H.; Herrmann, I.; Kunze, R.: Portius, H.J.: Schön, R., and Schönfeld, W.: Mechanism of digitalis action and the importance of the kinetics of the formation and decomposition of the glycoside-receptor complex for understanding of overall pharma cokinetics of digitalis compounds; in Storstein Symposium on digitalis, p. 143-151 (Gyldendal Norsk Eorlag, Oslo 1973). Schwiegk, H.: Die Therapie der Herzinsuffizienz mit Glykosiden. Internist. I: 6 -1 4 (1960). Takahashi, H.; Nakaya, S.: Takahashi, T. und Kalo, Y.: Über die minimalen Amplituden und die minimale Stillstandsdosis der Digitalisblätter und ihrer Präparate. 111. Versuch an Kaninchen. Tohoku J. exp. Med. 30: 85 97 (1936). Vassalle, M.: Karis, J., and Hoffman, B.F.: Toxic effects of ouabain on Purkinje fibers and ventricular muscle fibers. Am. J. Physiol. 203: 433 439 (1962). White, W.F. and Salter, W.T.: Response of hypodynamic myocardium to known concentra tions of cardiac glycosides. J. Pharmac. exp. Ther. 88: 1 -8(1946).
Different Toxic Effects of Ouabain and 16-Epi-Gitoxin on Purkinje Fibres and Ventricular Muscle Fibres Götz Nowak and Knut-Olaf Haustein Institute of Pharmacology and Toxicology, Medical Academy Erfurt, Erfurt
Key Words. Different actions on myocardium and Purkinje system • 16-Epi-gitoxin • Glycoside actions • Isolated Purkinje fibres • Isolated ventricular muscle fibres • Ouabain • Survival time of electrically driven cardiac fibres Abstract. Action potentials of isolated Purkinje fibres and ventricular muscle fibres of canine hearts treated with ouabain and 16-epi-gitoxin were recorded by microelectrodes. Under the influence of both glycosides, Purkinje system fibres became inexcitable earlier than ventricular muscle fibres. Being exposed to 0.\2S ijM ouabain and 2.5 yM 16-epigitoxin, both Purkinje and ventricular muscle fibres became poisoned in the same time as ventricular muscle fibres exposed to 0.1 tiM ouabain and 1.5 yM 16-epi-gitoxin. At the lower 16-epi-gitoxin concentration Purkinje fibres had 2.5 times the survival time of that exposed to the lower ouabain concentration. Compared to 16-epi-gitoxin the inexcitability of Purkinje fibres after ouabain remained irreversible. The semisynthetic glycoside 16-epigitoxin exerts a weaker effect on the specialized conducting system.
Based on a great number of experimental (Cat tell and Gold, 1941; Farah and Maresh, 1948; Gruhzit and Farah, 1953; Takahashi et at., 1936; White and Salter, 1946) and clinical studies (Batterman et al, 1952; Chavez, 1963; Schwiegk, 1960) it is supposed that there are no significant differences between therapeutic, toxic, and lethal doses of common digitalis preparations. However, in recent years it was shown that essential differences exist among natural glyco sides on the one hand (Ito et al, 1970) and between natural and semisynthetic glycosides on the other (Haustein et al., 1970; Haustein and Hauptmann, 1974). In order to obtain glycosides with a modified effect it seemed promising to utilize semisynthetic modifications of the genin moiety, an approach proven useful in other classes of drugs (glucocorticoids, anabolics and others). Comparative studies on 16-epi-gitoxin (Lindig and Repke, 1967), ouabain and gitoxin revealed that the effect of semisynthetic glycosides on excitable
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Received: September 25,1975.
Nowak ¡Haustein
257
tissues is much weaker than that of natural glycosides (Haustein el al, 1970). The investigations of Vassalle et al (1962) revealed that after exposure to oua bain Purkinje system fibres isolated from canine hearts were poisoned faster than ventricular muscle fibres. In the present experiments isolated Purkinje fibres and ventricular muscle fibres were incubated with ouabain and 16-epi-gitoxin to elucidate differences in their effects. The microelectrode technique according to Vassalle et al. (1962) was used for recording intracellular potentials. Starting from equieffective glycoside concentrations determined in the guinea pig heart concentrations are used producing a 30 % ( 0.1 hM ouabain, 1.5 juA/ 16-epi-gitoxin) or 50% (0.125 tiM ouabain, 2.5 ¡iM 16-epi-gitoxin) positive inotropic effect. Both ‘high’ glycoside concentrations led to an arrhyth mia frequency (At) 80 (ouabain) and 20% (16-epi-gitoxin), respectively, whereas after exposure to both ‘low’ glycoside concentrations only in the case of ouabain (50%) arrhythmias were observed. To study whether the toxic glyco side effects are reversible glycoside concentrations were used causing a fast maximum response (03 pM ouabain, 5.0 pM 16-epi-gitoxin). The arrhythmia frequency was 90 (ouabain) and 62 % (16-epi-gitoxin), respectively (Haustein et al, 1970). Materials and Methods Preparation Mongrel dogs of either sex between 1 and 3 years of age were anaesthetized with 25 mg sodium hexobarbital per kilogram body weight. After midsternal ihoracotomy the heart was removed, immediately washed in the nutritive medium, both ventricles opened, false ten dons and either trabeculae earneae or small papillary muscles rapidly removed. The prepara tions were transferred to 10 ml tissue baths and fixed with thin platinum electrodes, used for stimulation as well.
Electrophysiological Technique Hither of the two tissue baths contained one Purkinje or one ventricular preparation of the same canine heart. Stimulation was performed with square pulses < 1 2 msec, 0.2 I V) of an Orion-Vl.V function generator (Orion Electronics, Hungary). Action potentials were recorded by microclectrodcs according to Ling and Gerard (1949), filled with 3 M KCI. Tip diameters were less than 1 Mm and internal resistance was 7 10 Msi. The tissue baths were mounted on cross slides of two microscopes the ocular supports of which contained the cathode follow-up amplifiers (difference amplifier with field effect transistors, input resis tance 10M SI) with directly coupled electrodes. Both amplifiers were connected to a Mingograph 34-recorder (Elema-Schonander, Sweden) and a dual beam oscilloscope. Action potentials were recorded at a paper speed of 100 mm/sec. A three-axes stereomicroscope
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Composition o f the Nutritive Medium Nutritive medium composition was the following (in m/W): Na('l 137.0, KC1 5.5, MgC32 1.05, CaClj 2.7, NaHCOj 11.9, NaH2 PO., 1.78. glucose 5.55. The medium was matched to a pH of 7.2 with 0.1 N HC1, continuously bubbled with carbogen (95 % 0 2 /5 % C 02) and kept at 37 °C.
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fixed above the baths was used to observe the insertion of electrodes into the preparation. Action potentials of both preparations were taken from the third and fourth cell layers (passing the electrode through one cell layer abrupt increase of action potential amounting to 120 130 mV can be observed within it). Tissues were stimulated at a rate of 60/min. Recording Procedure After equilibration of 30 60 min the glycosides were added to the baths. Action potentials were recorded at 15-min intervals and mean values were calculated from 10 to 20 potentials, respectively. Duration and amplitude of action potentials were measured. The time interval between starting the glycoside exposure and the occurrence of inexcitability was referred to as survival time. Glycosides Ouabain according to DAB 6 (VEB Arzneimittelwerk, Dresden) and 16-epi-gitoxin (kindly supplied by the Central Institute of Molecular Biology, Academy of Sciences of GDR, Biomembrane Section, Berlin-Buch) were used. Both glycosides were dissolved in highly purified dimethylformamide, the 1 nuW stock solution was diluted with nutritive medium up to final concentration. The final concentration of dimethylformamide used throughout experiments was without any effect on fibre preparations.
Results
Effects o f 'Low ' Glycoside Concentrations on Ventricular Muscle and Purkinje Fibres (fig. 2, 3, table I) Both glycoside concentrations led to a 60 % decrease in action potential duration of ventricular muscle fibres and a 30 % (ouabain) and 50 % ( 16-epigitoxin) amplitude reduction, respectively. If Purkinje fibres are exposed to the above glycoside concentrations, differences in response result. Under the influ ence of ouabain, Purkinje fibres became inexcitable in about 85 min, as was also observed at higher concentration. Action potential duration dropped and the amplitude decreased to about the same values as with high ouabain concentra tion. In comparison, inexcitability appeared 200 min after starting 16-epi-gitoxin exposure. In this case, action potential duration dropped by about 35 % of
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Effects o f ‘High ’ Glycoside Concentrations on Ventricular Muscle and Purkinje Fibres (fig. 1, table I) Doses of 0.125 ¡M ouabain as well as of 2.5 juA/ 16-epi-gitoxin caused elec trical inexcitability of working muscle fibres within the same time. The duration of action potentials dropped about 60 % of the standard and the amplitude was about 50 % of the initial values. Purkinje fibres became inexcitable much faster than ventricular muscle fibres under both glycosides. The duration of action potentials dropped 30 % only, the amplitude, however, was reduced 50 (16-epigitoxin) and 60 % (ouabain), respectively. Ventricular muscle fibres had a sur vival time 2.1 or 2.3 times higher than Purkinje fibres.
259
Toxic Glycoside Effects on Isolated Cardiac Eibres
M inu te s
Fig. I. Effects of 0.125 nM ouabain () and 2.5 /wW 16-cpi-gitoxin (•) respectively on the amplitude (------ ) and duration (------ ) of action potentials of ventricular muscle fibres (A) or Purkinje fibres (B). The signs represent mean values of the experiments of table I.
Table I. Glycoside poisoning of isolated ventricular and Purkinje fibres of the canine heart Glycoside
Concentration
»M
Survival time (min ± SEM)
------------------ventricular muscle fibres
Ouabain
0.1
16-Epi-gitoxin
0.125 1.5 2.5
321 i 57(4) 186 ± 31(4) 343 ± 62(4) 156 ± 21 (4)
Purkinje fibres 81 89 200 68
? 11(5) i 21 (5) t 49 (5) ± 3(3)
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* The two values marked differ significantly (p < 0.001). Number of experiments indicated in parentheses.
Nowak/Haustein
260
M.nutes
Fig. 2. Effects of 0.1 tiM ouabain (o) and 1.5 uM 16-epi-gitoxin (•) respectively on the amplitude (------ ) and duration (------ ) of action potentials of ventricular muscle fibres (A) or Purkinje fibres (B). The signs represent the mean values of the experiments of table 1.
Reversibility o f Glycoside Effects ( Wash-Out Experiments) (fig. 4) Exposed to both glycoside concentrations, Purkinje fibres became inexcit able in 2 0 -3 0 min. After inexcitability occurred the preparation was washed three times with glycoside-free nutritive medium in 5 min. The ouabain inexcit ability remained irreversible for a further 60 min (fig. 4). In this time the ampli tude increased to 35 mV, additional wash-out procedures, however, did not eliminate toxic glycoside effects in the following 3 h. Compared to ouabain, Purkinje fibres poisoned with 16-epi-gitoxin gener ated first action potentials already 15 min after wash-out. The average amplitude
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initial values, the amplitude, however, decreased 75 %. Significantly lower ampli tudes were observed than with ouabain. Compared to ventricular muscle fibres, Purkinje fibres exposed to ouabain became poisoned 3.9 times, after 16-epigitoxin 1.7 times faster.
261
Toxic Glycoside Effects on Isolated Cardiac Fibres
01pA7 ouabain Ventricle fib re s
1.5
P urkinje fib re s
Ventricle fib re s
16-epi-gitoxm Purkinje fib re s
mm
J\_j Y l \ J \ IY J Y J V J \ !Y_A_ k _ _ K j\ J Y
------------
__ I?
o
J \ J \ , VJ v
i J \_ fY K J N 60
J \_ J V r v A
90
A __rv N _ K
120
A J V .tN _iN
ls e c 150
JV_A_
t\__ tv
180
J\__JV_
A_______ V,
2 *0
A _____ A .
r
/
300 0
JY JY
1 iY _ iV K _ K
Fig. 3. Effects of ouabain and 16-epi-gitoxin on action potentials of electrically driven ventricular muscle and Purkinje fibres of canine hearts in dependence on the time of glyco side incubation. Control: without glycoside incubation electrically driven fibre over the same time as within the incubation experiment.
increased to 93 mV and the duration to 450 msec 60 min after the initiation of this period. Thus, the initial values were nearly regained.
Our studies confirm the results of Vassalle et al. (1962) which revealed that Purkinje fibres of canine hearts became poisoned faster than ventricular fibres. If glycoside concentrations are causing a relatively high increase in contractile force
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Discussion
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(0.125/jA/ ouabain or 2.5 ju/W 16-epi-gitoxin) in the isolated guinea-pig heart, both glycoside concentrations showed no differences in toxic effects on canine hearts. Low concentrations of both glycosides (0.1 nM ouabain and 1.5 /jM 16epi-gitoxin) produced an equieffective moderate inotropic effect in the guineapig heart as well as the same time course of poisoning ventricular muscle fibres (table I). Considering both glycosides, marked differences in this time course were observed in Purkinje fibres. It could be shown that Purkinje fibres exposed to low glycoside concentrations had a survival time 2.5 times higher after 16epi-gitoxin than after ouabain (table 1; p < 0.001). Thus, the hypothesis stated in preceding studies is confirmed that the wider ‘therapeutic range' of 16-epi-
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Fig. 4. The influence of wash-out on poisoning Puikinje fibres with ouabain and 16epi-gitoxin. The amplitude and duration of action potentials restitute only after 16-epigitoxin. The spike potentials are stimulus artefacts as well as the reversed polarity occurring before action potential.
Toxic Glycoside Effects on Isolated Cardiac Fibres
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gitoxin is based on the weaker effect on the specialized conducting system (Haustein et al., 1970). The temporal differences in the glycoside effect occurrence could be explained by the results obtained in studying the kinetic parameters of the enzyme system (Repke et al., 1973). Accordingly, the delayed onset of Purkinje fibre inexcitability after 16-epi-gitoxin could be due to a much higher dissociation rate of the glycoside-receptor complex. In the (Na* + K+)-ATPase system of the guinea-pig heart, binding constants for the formation of glycosidereceptor complexes only slightly vary whereas the decay of the complex is 30 times higher in the case of 16-epi-gitoxin than in the case of ouabain (Repke etal., 1973). It is supposed that these results obtained with the receptor enzyme of the guinea-pig heart could be applied to the receptor of the Purkinje system. It is concluded that the glycoside-binding receptor areas of the (Na+ + K*)-ATPase of Purkinje and ventricular muscle fibres vary in conformation and possibly in structure. Differences in kinetics between the appearance of the effect and its disappearance or between complex formation and decay could be due to differ ent glycoside-binding receptors. Under the experimental conditions comparable to ours ,Pastelin and Mendez (1972) observed that poisoning of Purkinje fibres with 3|3-Z)-glucopyranosyl14/3-dihydroxy-2 l,23-bis-nor-5/3-chol-20(22)-ene-20-carboxylic acid 7 -lactone (AY 22241), a semisynthetic glycoside, was eliminated just as rapidly as in our studies on 16-epi-gitoxin. The results are not considered reliable because compar ative studies on ventricular muscle fibres or with other glycosides have not been performed yet. If the results obtained with isolated Purkinje and ventricular muscle fibres can be applied to the heart in situ, the following conclusions can be drawn to characterize differences in the effects of ouabain and 16-epi-gitoxin on the ventricular muscle fibre and Purkinje system: provided that the blood level of both glycosides decreases comparatively fast, the conducting system is more susceptible to the effects of ouabain than 16-epi-gitoxin. Action of the latter, however, is mainly directed to the working muscle. The presented results with 16-epi-gitoxin seem to support the hypothesis of Repke et al. (1970) that a wider range of inotropically effective glycoside concentrations following a moderate inhibition percentage of the ATPase system may be reached by semi synthetic modification of the genin moiety (Repke, 1970: Repke et al., 1973). Moreover, a dissociation of glycoside effects on working muscle and specialized conducting system is attained (Haustein et al., 1970).
The authors wish to thank Prof. /. Zell, Institute of Physiology of the Karl-MarxUnivcrsity Leipzig, for introduction to the electrophysiological technique, and Dipl.-lng. IP. Koth for the engineering of the amplifier equipment.
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A cknowledgements
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References
Dr. Knut-Olaf Haustein, Institut für Pharmakologie und Toxikologie, Medizinische Akade mie Erfurt, Nordhäuserstrasse 74, DDR 50 Erfurt (GDR)
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