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Influence of calcium on the action of Vipera ammodytes ammodytes snake venom on the myocardium
TO~dmn, Vol . 21, No. 6, pp. Printed in Grat Britain .
887-892, 1983 .
0041-OIOI/83 ß.00+ .00 ® 1983 Peraamon Preen Ltd.
INFLUENCE OF CALCIUM ON THE ACTION OF PIPERA AMMODYTES AMMODYTES SNAKE VENOM ON THE MYOCARDIUM DRAGOLJUB PETKOVIC,' MIRJANA PAVLOVI~,' DRAGICA MATEJEVI~, t NADEZDA UNKOVI~-CVETKOVIC, 3 TOMISLAV JOVANOVItr, t NADA ALEKSIC`,3 MIODRAG CVETKOVIC`,s ~ JELENA COLOVIC' and 130RIVOJE STAMENOVIC`t Departments of 'Physiology, 'Histology and 'Biochemistry, Faculty of Medicine, Belgrade, Yugoslavia (Accepted for publirntiort 12 April 1983) D. Psrtcovtt, M. PnvLOVIC, D. M~TFaEViC, N. UnrtcovtC-CvEtxovtC, T. JovwwovtC, N. AL~stC, M. G~rKOVit, J. ~OLOViâ.` and B. STnrreivovlC . Influence of calcium on the action of Yipera ammodytes arnnrodytes snake venom on the myocardium . Toxicon 21, 887 - 892, 1983 . - Y. arnmodytes menrodytes snake venom produced irreva~ble block of the isolated, artificially stimulated rat rightventricle or isolated rat heart. This was theresult of degenerative changes of the myocardium caused by thedirect effect of toxic components of the venom. An excess of Ca" could temporarily restart the contractions . PETKOVI~ et al. (1979) found that V. ammodytes ammodytes snake venom, as well as its toxic fractions, stopped the isolated rat heart in contracture, while the electrical activity persisted. This effect was the result of degenerative changes of myocardial cells caused by the action of toxic components present in the venom (Urtxovl~-CvETKOVIC et al., 1983). Proceeding from the fact that Cap* is very important for myocardial contractions, it was of interest to find out what changes would occur if the myocardium were exposed to an excess of Cap* in the presence of V. ammodytes ammodytes snake venom. Dried venom of Y. ammodytes ammodytes was obtained from the Institute of Immunology, Zagreb, Yugoslavia . Experiments were performed either on the isolated rat heart (PETKOVI~ et al., 1979) or on the isolated right ventricle of the rat heart. The ventricle was suspended in a bath containing 25 ml of oxygenated Tyrode solution of composition (mM) : NaCI 118; KC1 4.7; CaClz S; MgCh 4 .5; NaHCO, 25; glucose 5.6. Contractions were induced by electrical stimuli (1 Hz, twice threshold, 10 cosec) and recorded with an isometric transducer 7003 onto a microdynamometer ("Ugo 13asile" 7050, Varese, Italy) . After a stabilization period of 30 min, the crude venom or its fractions were added to the bath and contractions were recorded for 60 min. Also, the effects of the same concentrations of the crude venom or its fractions were investigated after the addition of 0.025 ml of CaCh (1 .8 mM) solution . CaCh was added either when the ventricle was quiescent after the application of the crude venom or its toxic fractions or at the same time with the crude venom or toxic fractions. After the addition of calcium into the bath, the contractions ~1'o whom requests for reprints should be addressed at : Department of Slochemiatry, Faculty of Medicine, Visegradaka 26, 11000 Belgrade, Yugoslavia.
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Short Commut»cations
were recorded for another 60 min and the ventricles were fixed for histological investigations . The material was fixed in 10% neutral formaldehyde and Carnoy solution, processed through paraffin, sectioned at S Nm and stained with haematoxylin - eosin according to the Azan method (BURCK, 1969). After the administration of 3 .75 mg of dissolved crude venom there was a significant decrease in the amplitude of contractions, which continued until the ventricle stopped contracting after about 12 min (Fig. 1, lower curve) . This effect was irreversible, even after washing the preparation for 1 hr. Histopathological studies on the myocardium showed a pronounced oedema with infiltration of cellular elements into the interstitial spaces . Certain muscle cells were oedematous and showed interruption of the sarcolemma (Fig . 2b). From 11 fractions obtained by the separation of the crude venom on Sephadex G-100, we used fractions F, and F6, because only these two fractions were toxic to the isolated rat heart (PETKOVI~ et al., 1979). Addition of 1 .43 mg of fraction F6 stopped the ventricle after an average of 20 min. The same effect was obtained when fraction F, (1 .10 mg) was applied and the ventricle stopped after an average of 22 min . Both effects were irreversible . Histopathological investigations showed similar changes as those caused by the whole venom. When the isolated right ventricle stopped contracting after the application of the crude venom or its toxic fractions (the venom or fractions were still present in the bath), CaCI, was added to the bath . Fifteen seconds after the addition of CaCI=, the ventricle restarted contracting and continued to contract for an average of 20 min in the presence of the whole venom (Fig . l, lower curve), 25 min with fraction F6 and 31 min with fraction F, . If CaCI, was added again, the ventricle started contracting once more and this process could be repeated several times, but the amplitude of contractions was always smaller with each new addition of CaCl z. The histopathological investigations showed parenchyma) degeneration of myocardial cells with a dim swelling of myofibrils and pycnotic nuclei (Fig . 2c).
FICA . l. EFFECTS OF' Vipera alrllg0(ly1PS SNAKE VEND\1 ON THE ISOLATED RAT RIGHT VENTRICLE . The lower curve represents the decrease in the tension after the addition of the crude venom to the bath . The arrow represents the addition of CaCI, which increases contraction tension . The upper curve represents simultaneous addition of the crude venom and CaCI, to the bath . n = 7.
FIG. i. HISTOPATHOLOGICAL INVESTIGATIONS OF THE MYOCARDIUM . (a) Control myocardium; (b) after application of the crude Vipers ammadytes snake venom; (c) after application of CaCI, when contractions stopped after application of the venom; (d) after simultaneous application of the venom and CaCI,. Scale 10 Nm .
Short Communications
89 1
Since the histopathological investigations showed changes which were more prominent in the presence of calcium than when the venom alone was applied, we performed experiments in which the ventricle was removed for histopathological investigations during the maximal effect of Ca" (4 min after Ca" ion application) . Only oedematous changes with infiltration of the interstitium and oedema in some muscle cells were observed, which is similar to effects of the venom alone. In some experiments, the bath was washed out when contractions stopped after venom administration and then CaClz was added . The contractions restarted again and continued for 1 hr after venom administration . The histopathological investigations of the myocardium showed similar changes as those after the action of the whole venom. When CaCI, was added to the bath at the same time as the crude venom or toxic fractions, a very significant increase in amplitude above the control level was observed . The amplitude then started to decrease slowly, but the contractions lasted much longer (42 min) than after the administration of the venom or the fractions alone (Fig . 1, upper curve) . The histopathological changes were more prominent than when the crude venom alone was applied. Besides the oedema of the whole myocardium there were pronounced degenerative changes in certain muscle cells . There was no cross-striation of myofibrils and indistinct muscle cells were found. In certain places the sarcolemma was interrupted. It was difficult to recognize cell nuclei and in some cells they were absent (karyolysis) (Fig. 2d). To confirm that these changes were not due to the effect of calcium application, we did some experiments in which only CaCh was used . The contractions were recorded for 1 hr and the ventricle was fixed for histopathological investigation, which showed only a slight oedema with no other morphological changes. Previously (PE~rxovl~ et al ., 1979), we found that the venom stopped the isolated perfused rat heart in the contracture state. Therefore, we decided to find out if injection of CaCI, into the cannula above the heart would restart it. When the heart stopped after injection of the venom, we injected CaCh and the heart restarted contracting, which continued to the end of the experiment (1 hr). If CaCI, was injected simultaneously with the venom, the amplitude of contractions increased immediately, then decreased slowly, but lasted to the end of the experiment (1 hr), The contracture which was manifested after the application of the venom alone did not appear . The histopathological investigations of the heart showed similar changes to those in the isolated right ventricle. V. ammodytes ammodytes venom irreversibly stopped the isolated artificially stimulated rat right ventricle. This was the result of degenerative changes in the myocardium caused by the direct effect of toxic fractions present in the venom, which was confirmed by the action of individual toxic fractions (UNKOVIi,`-CVETKOVIC et al., 1983). Similar effects were obtained with the venom of other snakes (LEE et al ., 1%8; Ho et al,, 1975 ; Znxl et al ., 1976). Our results are in agreement with the results of other authors, who found that an excess of calcium can antagonize nerve blocking and depolarizing effects of Naja naja atra venom (CI3ANG et al ., 1972) and the fibrillation of the heart produced by Ngja nigricolis venom (TAZIEFF-DEPIERRE et al., 1%9), and mitigate the blocking effects of both verapamil and disodium-EDTA on atrial contraction (Pnvl.ovl~ et al., 1979). The excess of calcium in the bath may stimulate the undamaged myofibrils and thereby restart contractions . At the same time, contractions of myofibrils may allow an increased penetration or effect of the venom, which will result in more severe degenerative changes.
892
Short Communications REFERENCES
BURCK, H. C. (1969) Physikalische and physiko-chemische Färbemethoden . In : Histologische Technik, p . 104, (2 . Auflage) . Stuttgart : Georg Thieme Verlag . CHANG, C. C., CHUANG, SING-TAI, LEE, C. Y. aRd WEI, .I . W. (1972) Role of cardiotoxin and phospholipase A in the blockade of nerve conduction and depolarization of skeletal muscle induced by cobra venom. Br. J. Pharmac. 44, 732. Ho, Ch . L., LEE, C. Y . and Lu, H. H. (1973) Electrophysiological effects of cobra cardiotoxin on rabbit heart cells. Toxicon 13, 437. LEE, C. Y., CHANG, C. C ., CHIN, T. H ., CHIN, P . J . S., TSEN(G, T. C . and LEE, S . Y. (1968) Pharmacological properties of cardiotoxin isolated from Formosan cobra venom. Arch . ezp. Path . Pharmak. 259, 360. PAVt.ovIC, M. R., MATErEVIC, D. R. and VAxAGIt, V. M. (1979) The influence of Ca*' antagonists on the inotropic effect of dibutyryl cyclic AMP on the isolated atria of the rat . lug. Physiol. Pharmac. Acts 15, 123 . PETKOVIt, D., JUV.ANOVIC, T., MItEVIr, D., UNKOVIC-CVETKOVIC, N. and Cv~KOVit, M. (1979) Action of Vipers ammodytes ammodytes venom and its fractions on the isolated rat heart . Toxicon 17, 639. TAZtEeF-DI3P~ItRE, F., CZAJKA, M. and LOWAGIE, C. (1969) Action pharmacologique de fractions puree de venin de Ngja nigricollis et libération de calcium dens lee muscles stri&. C. r. hebd. Séwrc. Acad. Sci., Paris 268, 2311 . LINKOVIC-CVETKOVIt`, N., CVETKOVIC, M., PETKOVIC, D., JOVANOVIt`, T. SRd UNKOVIC, S. (1983) Histopathological changes of rat myocardium caused by Vipers ammodytes ammodytes (European viper) snake venom. Toxicon 21, 429. ZAKI, A. O., HANNA, N., NEN'AT, B., KAJUaI, S. and PETKOVIC, D. (1976) Circulatory effect of Bitis gabonica venom. Ain Shams Med. J. 27, 133 .
887-892, 1983 .
0041-OIOI/83 ß.00+ .00 ® 1983 Peraamon Preen Ltd.
INFLUENCE OF CALCIUM ON THE ACTION OF PIPERA AMMODYTES AMMODYTES SNAKE VENOM ON THE MYOCARDIUM DRAGOLJUB PETKOVIC,' MIRJANA PAVLOVI~,' DRAGICA MATEJEVI~, t NADEZDA UNKOVI~-CVETKOVIC, 3 TOMISLAV JOVANOVItr, t NADA ALEKSIC`,3 MIODRAG CVETKOVIC`,s ~ JELENA COLOVIC' and 130RIVOJE STAMENOVIC`t Departments of 'Physiology, 'Histology and 'Biochemistry, Faculty of Medicine, Belgrade, Yugoslavia (Accepted for publirntiort 12 April 1983) D. Psrtcovtt, M. PnvLOVIC, D. M~TFaEViC, N. UnrtcovtC-CvEtxovtC, T. JovwwovtC, N. AL~stC, M. G~rKOVit, J. ~OLOViâ.` and B. STnrreivovlC . Influence of calcium on the action of Yipera ammodytes arnnrodytes snake venom on the myocardium . Toxicon 21, 887 - 892, 1983 . - Y. arnmodytes menrodytes snake venom produced irreva~ble block of the isolated, artificially stimulated rat rightventricle or isolated rat heart. This was theresult of degenerative changes of the myocardium caused by thedirect effect of toxic components of the venom. An excess of Ca" could temporarily restart the contractions . PETKOVI~ et al. (1979) found that V. ammodytes ammodytes snake venom, as well as its toxic fractions, stopped the isolated rat heart in contracture, while the electrical activity persisted. This effect was the result of degenerative changes of myocardial cells caused by the action of toxic components present in the venom (Urtxovl~-CvETKOVIC et al., 1983). Proceeding from the fact that Cap* is very important for myocardial contractions, it was of interest to find out what changes would occur if the myocardium were exposed to an excess of Cap* in the presence of V. ammodytes ammodytes snake venom. Dried venom of Y. ammodytes ammodytes was obtained from the Institute of Immunology, Zagreb, Yugoslavia . Experiments were performed either on the isolated rat heart (PETKOVI~ et al., 1979) or on the isolated right ventricle of the rat heart. The ventricle was suspended in a bath containing 25 ml of oxygenated Tyrode solution of composition (mM) : NaCI 118; KC1 4.7; CaClz S; MgCh 4 .5; NaHCO, 25; glucose 5.6. Contractions were induced by electrical stimuli (1 Hz, twice threshold, 10 cosec) and recorded with an isometric transducer 7003 onto a microdynamometer ("Ugo 13asile" 7050, Varese, Italy) . After a stabilization period of 30 min, the crude venom or its fractions were added to the bath and contractions were recorded for 60 min. Also, the effects of the same concentrations of the crude venom or its fractions were investigated after the addition of 0.025 ml of CaCh (1 .8 mM) solution . CaCh was added either when the ventricle was quiescent after the application of the crude venom or its toxic fractions or at the same time with the crude venom or toxic fractions. After the addition of calcium into the bath, the contractions ~1'o whom requests for reprints should be addressed at : Department of Slochemiatry, Faculty of Medicine, Visegradaka 26, 11000 Belgrade, Yugoslavia.
888
Short Commut»cations
were recorded for another 60 min and the ventricles were fixed for histological investigations . The material was fixed in 10% neutral formaldehyde and Carnoy solution, processed through paraffin, sectioned at S Nm and stained with haematoxylin - eosin according to the Azan method (BURCK, 1969). After the administration of 3 .75 mg of dissolved crude venom there was a significant decrease in the amplitude of contractions, which continued until the ventricle stopped contracting after about 12 min (Fig. 1, lower curve) . This effect was irreversible, even after washing the preparation for 1 hr. Histopathological studies on the myocardium showed a pronounced oedema with infiltration of cellular elements into the interstitial spaces . Certain muscle cells were oedematous and showed interruption of the sarcolemma (Fig . 2b). From 11 fractions obtained by the separation of the crude venom on Sephadex G-100, we used fractions F, and F6, because only these two fractions were toxic to the isolated rat heart (PETKOVI~ et al., 1979). Addition of 1 .43 mg of fraction F6 stopped the ventricle after an average of 20 min. The same effect was obtained when fraction F, (1 .10 mg) was applied and the ventricle stopped after an average of 22 min . Both effects were irreversible . Histopathological investigations showed similar changes as those caused by the whole venom. When the isolated right ventricle stopped contracting after the application of the crude venom or its toxic fractions (the venom or fractions were still present in the bath), CaCI, was added to the bath . Fifteen seconds after the addition of CaCI=, the ventricle restarted contracting and continued to contract for an average of 20 min in the presence of the whole venom (Fig . l, lower curve), 25 min with fraction F6 and 31 min with fraction F, . If CaCI, was added again, the ventricle started contracting once more and this process could be repeated several times, but the amplitude of contractions was always smaller with each new addition of CaCl z. The histopathological investigations showed parenchyma) degeneration of myocardial cells with a dim swelling of myofibrils and pycnotic nuclei (Fig . 2c).
FICA . l. EFFECTS OF' Vipera alrllg0(ly1PS SNAKE VEND\1 ON THE ISOLATED RAT RIGHT VENTRICLE . The lower curve represents the decrease in the tension after the addition of the crude venom to the bath . The arrow represents the addition of CaCI, which increases contraction tension . The upper curve represents simultaneous addition of the crude venom and CaCI, to the bath . n = 7.
FIG. i. HISTOPATHOLOGICAL INVESTIGATIONS OF THE MYOCARDIUM . (a) Control myocardium; (b) after application of the crude Vipers ammadytes snake venom; (c) after application of CaCI, when contractions stopped after application of the venom; (d) after simultaneous application of the venom and CaCI,. Scale 10 Nm .
Short Communications
89 1
Since the histopathological investigations showed changes which were more prominent in the presence of calcium than when the venom alone was applied, we performed experiments in which the ventricle was removed for histopathological investigations during the maximal effect of Ca" (4 min after Ca" ion application) . Only oedematous changes with infiltration of the interstitium and oedema in some muscle cells were observed, which is similar to effects of the venom alone. In some experiments, the bath was washed out when contractions stopped after venom administration and then CaClz was added . The contractions restarted again and continued for 1 hr after venom administration . The histopathological investigations of the myocardium showed similar changes as those after the action of the whole venom. When CaCI, was added to the bath at the same time as the crude venom or toxic fractions, a very significant increase in amplitude above the control level was observed . The amplitude then started to decrease slowly, but the contractions lasted much longer (42 min) than after the administration of the venom or the fractions alone (Fig . 1, upper curve) . The histopathological changes were more prominent than when the crude venom alone was applied. Besides the oedema of the whole myocardium there were pronounced degenerative changes in certain muscle cells . There was no cross-striation of myofibrils and indistinct muscle cells were found. In certain places the sarcolemma was interrupted. It was difficult to recognize cell nuclei and in some cells they were absent (karyolysis) (Fig. 2d). To confirm that these changes were not due to the effect of calcium application, we did some experiments in which only CaCh was used . The contractions were recorded for 1 hr and the ventricle was fixed for histopathological investigation, which showed only a slight oedema with no other morphological changes. Previously (PE~rxovl~ et al ., 1979), we found that the venom stopped the isolated perfused rat heart in the contracture state. Therefore, we decided to find out if injection of CaCI, into the cannula above the heart would restart it. When the heart stopped after injection of the venom, we injected CaCh and the heart restarted contracting, which continued to the end of the experiment (1 hr). If CaCI, was injected simultaneously with the venom, the amplitude of contractions increased immediately, then decreased slowly, but lasted to the end of the experiment (1 hr), The contracture which was manifested after the application of the venom alone did not appear . The histopathological investigations of the heart showed similar changes to those in the isolated right ventricle. V. ammodytes ammodytes venom irreversibly stopped the isolated artificially stimulated rat right ventricle. This was the result of degenerative changes in the myocardium caused by the direct effect of toxic fractions present in the venom, which was confirmed by the action of individual toxic fractions (UNKOVIi,`-CVETKOVIC et al., 1983). Similar effects were obtained with the venom of other snakes (LEE et al ., 1%8; Ho et al,, 1975 ; Znxl et al ., 1976). Our results are in agreement with the results of other authors, who found that an excess of calcium can antagonize nerve blocking and depolarizing effects of Naja naja atra venom (CI3ANG et al ., 1972) and the fibrillation of the heart produced by Ngja nigricolis venom (TAZIEFF-DEPIERRE et al., 1%9), and mitigate the blocking effects of both verapamil and disodium-EDTA on atrial contraction (Pnvl.ovl~ et al., 1979). The excess of calcium in the bath may stimulate the undamaged myofibrils and thereby restart contractions . At the same time, contractions of myofibrils may allow an increased penetration or effect of the venom, which will result in more severe degenerative changes.
892
Short Communications REFERENCES
BURCK, H. C. (1969) Physikalische and physiko-chemische Färbemethoden . In : Histologische Technik, p . 104, (2 . Auflage) . Stuttgart : Georg Thieme Verlag . CHANG, C. C., CHUANG, SING-TAI, LEE, C. Y. aRd WEI, .I . W. (1972) Role of cardiotoxin and phospholipase A in the blockade of nerve conduction and depolarization of skeletal muscle induced by cobra venom. Br. J. Pharmac. 44, 732. Ho, Ch . L., LEE, C. Y . and Lu, H. H. (1973) Electrophysiological effects of cobra cardiotoxin on rabbit heart cells. Toxicon 13, 437. LEE, C. Y., CHANG, C. C ., CHIN, T. H ., CHIN, P . J . S., TSEN(G, T. C . and LEE, S . Y. (1968) Pharmacological properties of cardiotoxin isolated from Formosan cobra venom. Arch . ezp. Path . Pharmak. 259, 360. PAVt.ovIC, M. R., MATErEVIC, D. R. and VAxAGIt, V. M. (1979) The influence of Ca*' antagonists on the inotropic effect of dibutyryl cyclic AMP on the isolated atria of the rat . lug. Physiol. Pharmac. Acts 15, 123 . PETKOVIt, D., JUV.ANOVIC, T., MItEVIr, D., UNKOVIC-CVETKOVIC, N. and Cv~KOVit, M. (1979) Action of Vipers ammodytes ammodytes venom and its fractions on the isolated rat heart . Toxicon 17, 639. TAZtEeF-DI3P~ItRE, F., CZAJKA, M. and LOWAGIE, C. (1969) Action pharmacologique de fractions puree de venin de Ngja nigricollis et libération de calcium dens lee muscles stri&. C. r. hebd. Séwrc. Acad. Sci., Paris 268, 2311 . LINKOVIC-CVETKOVIt`, N., CVETKOVIC, M., PETKOVIC, D., JOVANOVIt`, T. SRd UNKOVIC, S. (1983) Histopathological changes of rat myocardium caused by Vipers ammodytes ammodytes (European viper) snake venom. Toxicon 21, 429. ZAKI, A. O., HANNA, N., NEN'AT, B., KAJUaI, S. and PETKOVIC, D. (1976) Circulatory effect of Bitis gabonica venom. Ain Shams Med. J. 27, 133 .