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Mechanism of action of purified scorpion toxin on the isolated rat intestine
Toxlcon, 1973, Vol. 11, pp . 81-84. Pat;emon Press. Printed in Great Hritein
MECHANISM OF ACTION OF PURIFIED SCORPION TOXIN ON THE ISOLATED RAT INTESTINE* J. R. Ctnvxn MEw,t L. FREIRE-MAiw,t W. L. TAr-IrRlt and T. A. MARIAt Department of Physiology, Institute of Biological Sciences, UFMG, CP. 2486, and Department of Pathology, Faculty of Medicine, UFMG, Belo Horizonte, MG. Brazil (Acceptedjorpublication 101uly 1972)
Abstract-PuriSed scorpion toxin (Tityus serrulatus) produced contraction of the isolated rat ileum, followed by an increase in the amplitude of pendular movement and rhythmic variation of tonus. These effects may be due to the release of acetylcholine and substance P. Toxin praduoed relaxation in the atropinized rat duodenum, which was abolished by alpha and beta sympatholytic drugs. The contraction produced by S-HT in the rat ileum was either decreased or abolished by toxin . In the toxin-treated ileum, nicotine produced only relaxation. The mechanisms of the inhibitory effects of toxin on the contractions produced by S-HT and nicotine are discussed . The relaxation produced by nicotine was abolished by alpha and beta sympatholytic drugs and by ganglionic blocidng agents . INTRODUCTION 1~ECTS of crude scorpion venoms have been studied on the isolated guinea-pig and rat ileum (D>~, Pozo, 1956 ; D1Nlz and Goxç~LVlE;.s, 1956; PAZ-rntsoN, 1962) . Our understanding of the venom actions still remains incomplete and it was decided to investigate the subject using a purified fraction of the venom of Brazilian scorpion Tltyus serrulatus. Preliminary reports of our results have been presented elsewhere (CuNxA MEr o et al., 1970 ; FytE1REMAIn and Dl:xlz, 1970) . T1IS
METHODS The rat intestine was suspended in 10 ml aerated Jalon solution (35°C) which had the following composition : sodium chloride 9 g; potassium chloride 042 g ; calcium chloride 006 g ; sodium bicarbonate 0~5 g; glucose 0~5 g; sufficient deionized water to make one liter (BURN et al., 1950) . The contractions were recorded on a smoked paper on a kymograph by means of a frontal writing lever. The agonistic substances were added to the organ bath in a sub-maximal dosage, remaining in contact with the muscle for 30-60 sec and then washed out. Antagonists and toxin were allowed to act for periods of up to 5 min . Drugs used : acetylcholine chloride (Roche), adrenaline hydrochloride (Parke-Davis), atropine sulphate (Sigma), cocaine hydrochloride (Merck), physostigmine (eserine) sulphate (Merck), pentolinium tartrate (Ansolysen,® Wyeth), hexamethonium chloride (Squibb), methysergide maleate (Sandoz), phentolamine mesylate (Ciba), propranolol (Inderal,® Ayerst), 5-hydroxytryptamine creatinine sulphate (serotonin, Koch-Light), nicotine (Sigma), 'This paper was supported by Conselho National de Pesquisas and Conselho de Pesquisas da UFMG., Brazil. tFellows of CNPq., Brazil. 81
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bradykinin (Sandoz, BRS-640, Batch no. 69055) and substance P.* Tityus serrulatus venom was obtained from Instituto Butantan (Sâo Paulo, Brazil) and purified toxin fraction (T~ was prepared usinga combination of precipitation and column chromatography on sephadex G-25 (GOMFZ and DtNIZ, 1966) . Samples of purified toxin, kindly provided by Drs. M. V. Gomez and C. R. Diniz, from the Dept. of Biochemistry, 1CB, Belo Horizonte, Brazil, are devoid of phospholipase A and other enzymatic activities . The quantities of toxin are expressed as weights ofprotein . RESULTS
E ~ect of toxin on the rat ileum. When toxin (1-5 tag/ml) was added to the organ bath containing rat ileum, the muscle contracted 3-5 cm. In this preparation acetylcholine (5~5 x 10-8 to 1 ~1 x 10-'M), bradykinin (4~7 x 10 - e to 9-4 x 10-°M) and 5-HT (6~4 x 10-' to 2~6 x 10-°M) produced equiactive contractions (20 exp). The contraction induced by toxin is of a slow type, starting after a latent interval ranging from 10 to 60 sec, thereby differing from that produced by acetylcholine, bradykinin and 5-HT (Fig. 1 A). Following the slow response the muscle showed spasmodic contractures which lasted for 2 to 4 hr, even after repeated washings with Jalon solution . However, addition of atropine to the organ bath (1 ~5 x 10-'M) brought the muscle back to its original tone. In 10 out of 20 experiments a reinforcement of pendular movements and appearance of rhythmic contractions were also observed (Fig. 1A). Subsequent doses of toxin produced responses of smaller magnitude or no response at all (tachyphylaxis). Fig. 1B shows one of these experiments . On the other hand, larger doses of toxin (10 tcg/ml) produced fast contractions which returned to the base line 5-10 min after washing out the preparation (20 exp). In 10 experiments a relaxation was observed after the second addition of toxin ; the latter effect is not tachyphylactic, since it can be repeated by subsequent additions of the same doses of toxin . Mode ofaction oftoxin on the rat ileum. In order to determine the possible participation of acetylcholine in the mechanism of toxin action the rat ileum was suspended in a Jalon solution containing atropine (1 ~5 x 10-'M) . Under these circumstances, the addition of toxin to the bath (5-10 Fig/ml) produced in 8 out of 16 experiments a small relaxation which preceded the contraction (Fig. 2A). The reinforcement of pendular movement and rhythmic contractions were also recorded after addition of toxin to atropinized preparations. On the other hand, the addition of eserine to the bath (1 ~5 x 10-~M) potentiated the contraction produced by toxin (1 t`g/ml) ; moreover, subliminar doses of toxin (0~1 ~g/ml) produced contraction of the eserine treated preparations . Eserine was able to increase the muscular spasm produced by a previous dose of toxin (1 S exp). Two ofthese experiments are shown in Fig. 2, B and C. Ganglionic blockade with pentolinium (1 ~8 x 10-6M) or hexamethonium (3 ~7 x I O-°M) failed to prevent contractions produced by toxin (1-5 tag/ml, 5 exp). These results agree with those reported by DtrIIZ and GorrçALVES (1956) in the guinea-pig ileum. Serotonin (2~6 x 10-' to 2~6 x 10-°M) when added to an ileum segment which had previously received toxin (1 to 5 tag/ml), either failed to contract the preparation or evoked contractions smaller than those obtained before toxin (16 exp). Methysergide (1 ~1 x 10-°M) blocked the effects of Serotonin on the ileum, but had no influence on the action of toxin (7 exp). Atropine and methysergide when added simultaneously to the bath abolished the effects of acetylcholine "Kindly provided by Prof. F. Lembeek, from the Institute of Pharmacology, University of Graz, Universitätsplatz 4, A-ß0I0 Graz, Austria. TOXICON J973 YoJ. JJ .
FIG. 1 . RAT ILEUM PREPARATION SUSPENDED IN 1O ml JALON SOLUTION . Acetylcholine (ACh). 1 ~1 x 10 'M ; bradykinin (Bk), 9~4 r 10 -°M ; serotonin (5-HT), 6~4 x l0-'M and purified scorpion toxin (T), 3 ~g/ml. After each addition to the bath the preparation was rinsed once or twice ( 1 1 ). In tracing B, a period of 30 min elapsed between thefirstand the second addition of toxin ('T) .
FIG. 2. RAT ILEUM PREPARATIONS SUSPENDED IN IO ml JALON SOLUTION .
Tracing A. Acetylcholine (ACh), 2~7 :~ 10 -~M ; bradykinin (Bk), 1~9 : : 10-°M and purified scorpion toxin (T), 10 Fig/ml ; atropine (1~5 " 10-'M) was present in the bath throughout . Tracing B. Acetylcholine (ACh), 1~1 ~ 10 -"M ; toxin (T), 0~1 l+g/ml and eserine (E), I~5 ï": 10-'M . Tracing C. Toxin (T), 1 Fig/ml ; acetylcholine and eserine, as in B. After each addition to the bath the preparation was rinsed once or twice ( 1 1 ). roxfcoN ~ms v~r. m.
f.p . 82
FIG. 3. RAT ILEUM PREPARATIONS SUSPENDED IN lO mI JALON SOLUTION . Tracing A. Acetylcholine (ACh), 1~1 ": 10- 'M ; serotonin (5-HT), 7-8 >: 10 - ~M and purified scorpion toxin ('~, 2 pg/ml. A period of 30 min elapsed between the additions of toxin and 5-HT. Tracing B. Acetylcholine (ACh), 1 ~1 a ]0-'M ; serotonin (5-HT), 2~6 x 10-'M and purified scorpion toxin (T), 5 ~g/ml. At larger arrow, atropine (1 ~S x 10 -'M) and methysergide (1 ~1 >~ 10-"M) were added to the bath . ! 1 represents rinsing of the preparation .
TOXICON 1973 Vol. 11 .
FIG. 4. RAT ILEUM PREPARATION SUSPENDED IN IO ml .TALON SOLUTION . Adrenaline (Ad), 2~3 x 10 -'M ; nicotine (Nic), I~5 x 10-"M ; acetylcholine (ACh), 5~5 f 10 -°M and purified scorpion toxin (T), 10 l+g/ml . After each addition, the preparation was rinsed once or twice ( 1 1 ). A period of 15 min elapsed between the first dose of toxin and the second dose of adrenaline. In tracing B, phentolamine (2~6 x 10-°M) and propranolol (1 ~7 :< 10-"M) were maintained in the bath throughout the experiments.
FIG. S . RAT DUODENUM PREPARATIONS SUSPENDED IN IO ml JALON 30LUTION, Tracings A and B, atropine (I ~5 :~: 10- 'M) was maintained in the bath throughout the expts Adrenaline (Ad), 2~3 x 10- 'M ; bradykinin (Bk), 9~4 :~: ]0 -°M ; acetylcholine (ACh), 5 ~5 ~ 10-'M and purified scorpion toxin (T), 21~g/ml. In tracing B, phentolamine (2~6 ~ 10-°M) and propranolol (1 ~7 :< 10-°M) were present in the bath throughout the expts. At arrows, the bath fluid was changed. TOXICON 1973 Vol. 11 .
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and serotonin but did not prevent the contraction produced by toxin (6 exp) . Two of these experiments are shown in Fig. 3, A and B. Cocaine (7~4 x 10 -6 M) prevented the contraction produced by toxin (1-5 Fig/ml), but had no effect on the response caused by acetylcholine (5~5 x l0-e to 1 ~1 x 10-'M, 6 exp) . These results also agree with those described by Dirnz and Goxçnr vi:.s (1956) in the guinea-pig ileum. Propranolol (1 ~7 x 10-6 M) and phentolamine (2.6 x 10-bIV17 prevented the relaxation produced by toxin (10 hg/ml) and adrenaline (2 ~3 x 10-'M, 6 exp) . The results of one of these experiments are shownin Fig. 4. Addition of nicotine to the bath (6~2 x 10-8 to 1 ~5 x 10 -bM) caused contraction of the ileum. After toxin, the same dose of nicotine produced relaxation (20 exp), which was prevented either by propranolol and phentolamine (Fig . 4) or pentolinium (1 ~8 x 10 -°M). Reinforcement of pendular movements and rhythmic contractions caused by toxin were similar to those produced by substance P (0~5 mg/ml) . E ~ect of toxin on the rat duodenum . Addition of toxin to the rat duodenum (1-5 hg/ml) suspended in atropinized Jalon solution produced relaxation of the intestine (20 exp) . This effect was abolished by propranolol and phentolamine added simultaneously to the bath, before toxin (Fig . ~. As in the ileum, toxin caused contraction and increased the amplitude of pendular movements of the non-atropinized duodenum (8 exp) . DISCUSSION Isolated smooth muscle contractions caused by scorpion venom (Tityus serrulatus) have been claimed to be due to the release of an acetylcholine-like substance (Dixlz and GONÇALVFS, 1956 ; Dmnz and Vnr mi, 1959 ; Dnviz and ToxRES, 1968). In the present experiments, however, it was shown that rat ileum contractions produced by purified scorpion toxin were only partially blocked by atropine. As the contractions were not blocked by methysergide, the participation of 5-HT on the mechanism of contraction produced by toxin can be ruled out. The enhancement of intestinal tonus could be due to a continuous release of a mediator or mediators which cause a prolonged state of contraction . One of these mediators probably is acetylcholine, since eserine increased and atropine decreased the muscular spasm. In addition, the reinforcement of pendular movement and rhythmic variations of tonus caused by toxin, even in atropinized preparations, could be due to release of substance P. This hypothesis is supported by experiments showing reduction in the substance P content of rat gut after injection of scorpion toxin (HIAL and Drnnz, 1971). The tachyphylaxis observed after toxin could be due to inability of the ileum to release active substance (s), for the actions of acetylcholine and substance P were not abolished. On the other hand, the reduced effect shown by serotonin after toxin could be ascribed to the blockade of M receptors (GADDUM and PICARELLI, 1957) ; however, the number of these receptors in rat gut seems to be very small (LEVY and Mtet~,-B~t, 1956 ; GARATI'INI and VALZELLI, 196 . As substance P is able to block the contraction produced by serotonin (BELESLIN and VAR.AGIC, 1960), it is suggested that inhibition of the effect of 5-HT is related to the release of substance P from the intestine by scorpion toxin. Another possibility is that toxin blocks D receptors. The relaxation induced by toxin depends on an adrenergic mechanism, since alpha and beta sympatholytic drugs abolished it. These experiments are in accordance with a previous paper from this laboratory, which shows that the hypertensive effect of toxin in anesthetized rats is due to release of catecholamines (FytEIRE-MAm and DINIZ, 1970). The inversion of the 710XICON 1973 Yol. ll . E
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nicotine effect produced by scorpion toxin ~in the rat ileum is similar to that described by AMSncxe (1951) in the rabbit ileum treated with botulinum toxin . It seems that scorpion toxin blocks the release of acetylcholine caused by nicotine . In addition, since the relaxation caused by nicotine is abolished both by ganglionic blockade and sympatholytic drugs it seems likely that it is due to release of catecholamines . Scorpion toxin causes a decrease in the number of the granular vesicles and a simultaneous increase in the agranular vesicles in the myenteric plexus of the rat (TAFIJRI et al., 1971) . It seems likely that the reduction in the number of the granular vesicles could be related to the release ofcatecholamines and perhaps substance P. Acknowledgement-The Authors express their sincere appreciation to Prof. W. T. BERALDO for helpful criticism of the manuscript and Mr . José Lutz Dw Cosrw for technical assistance . REFERENCES A~wcEtt:, N. (1951) Unmasking, after cholinergic paralysis by botulinum toxin, of a reversed action of nicotine on the mammalian intestine, revealing the probable presence of local inhibitory ganglion cells in the enteric plexuses. Br. J. Pharmac. 6, Sl . BELe4LIN, D. and Vwxwctc, V. (1960) The effort of substance P on the responses of the isolated guinea-pig ileum to acetylcholine, nicotine, histamine and 5-hydroxytryptamine. Archs int. Pharmacodyn. Thér. 126, 321 . Battra, J. H., Ftxrvt:v, D.J. and Goonwnv, L. G. (1950) Biological standardization, Sec . edition, p. 223 . London Oxford University Press. CtJxttw Mta.O, J. R, FRF14F-Mwu, L., TAFUttt, W. L. and Mwnu, T. A. (1970) Mecanismo de açito da toxins purificada de esoorpillo sobre o 11eo isolado de rato . Acrd physiol. latinoam . 20, 296. DEt, Pozo, E. D. (1956) Mechanism of pharmacological actions of scorpion venoms . In : Venoms (BUCKLEY, E. E. and POxaFS, N., Eds.), p. 123. Washington : Am . Ass. Adv. Sri. Dttatz, C. R. and Gotaçwt.ves, J. M. (1956) Some chemical and pharmacological properties of Brazilian scorpion venoms. In : Venoms (BuctcLEV, E. E. and Pondes, N., Eds.), p. 131 . Washington : Am . Ass. Adv. Sci. Dtraz, C. R. and Vwt.sw, V. (1959) Effects of a toxin present in a purified extract of telsons from thescorpion Titvus serrulatus on smooth muscle preparations and in mice. Archs inr. Pharmacodyn . Thér. 121, 1 . Dnvtz, C. R. and Totuees, J. M. (1968) Release of an acetylcholine-like substance from guinea-pig ileum by scorpion venom. Toxlcon 5, 277. Ftt~-Mwu, L. and Dttatz, C. R. (1970) Pharmacological action of a purified scorpion toxin in the rat. Toxlcon 8, 132. GADDUM, J. H. and Ptcwxia.u, Z. P. (1957) Two kinds of tryptamine receptor . Br . J. Pharmac. 12, 323. Gwrcwrnru, S. and Vwtzaru, L. (1965) Seroronin, p. 87 . Amsterdam: Elsevier . Go~z, M . V. and Dtrrtz, C. R. (1966) Separation of toxic components from the Brazilian scorpion-Tiryus serrulatus-venom. Mrnrs Isst . Butantan 33, 899. Hut., W. and Dnatz, C. R. (1971) Efeito da scorpiotoxina sobre o conteudo de substltncia P do intestino de rato . Ciénc. Cult ., S. Paulo, 23 (supl), 304. LEVY, J. and Mtcttet.-Brat, E. (1956) . Contribution a l'action pharmacologique exercée par la sdrotonine sur quelques organes isolés (intestin et oreillette). J. Physlol., Paris 48. 1051 . Pwrreagorr, R. A. (1962) Pharmacologie action of scorpion venom on intestinal smooth muscle. Toxic. rrppl. Pharmac. 4, 710. Tw~ttt, W. L., Mwww, T. A., FREIRE-MAIA, L. and C~trattw MEt.o, J. R. (1971) Effect of purified scorpion toxin on vesicular components in the myenteric plexus of the rat. Toxlcon 9, 427.
770XICON 1973 Vol. tl .
MECHANISM OF ACTION OF PURIFIED SCORPION TOXIN ON THE ISOLATED RAT INTESTINE* J. R. Ctnvxn MEw,t L. FREIRE-MAiw,t W. L. TAr-IrRlt and T. A. MARIAt Department of Physiology, Institute of Biological Sciences, UFMG, CP. 2486, and Department of Pathology, Faculty of Medicine, UFMG, Belo Horizonte, MG. Brazil (Acceptedjorpublication 101uly 1972)
Abstract-PuriSed scorpion toxin (Tityus serrulatus) produced contraction of the isolated rat ileum, followed by an increase in the amplitude of pendular movement and rhythmic variation of tonus. These effects may be due to the release of acetylcholine and substance P. Toxin praduoed relaxation in the atropinized rat duodenum, which was abolished by alpha and beta sympatholytic drugs. The contraction produced by S-HT in the rat ileum was either decreased or abolished by toxin . In the toxin-treated ileum, nicotine produced only relaxation. The mechanisms of the inhibitory effects of toxin on the contractions produced by S-HT and nicotine are discussed . The relaxation produced by nicotine was abolished by alpha and beta sympatholytic drugs and by ganglionic blocidng agents . INTRODUCTION 1~ECTS of crude scorpion venoms have been studied on the isolated guinea-pig and rat ileum (D>~, Pozo, 1956 ; D1Nlz and Goxç~LVlE;.s, 1956; PAZ-rntsoN, 1962) . Our understanding of the venom actions still remains incomplete and it was decided to investigate the subject using a purified fraction of the venom of Brazilian scorpion Tltyus serrulatus. Preliminary reports of our results have been presented elsewhere (CuNxA MEr o et al., 1970 ; FytE1REMAIn and Dl:xlz, 1970) . T1IS
METHODS The rat intestine was suspended in 10 ml aerated Jalon solution (35°C) which had the following composition : sodium chloride 9 g; potassium chloride 042 g ; calcium chloride 006 g ; sodium bicarbonate 0~5 g; glucose 0~5 g; sufficient deionized water to make one liter (BURN et al., 1950) . The contractions were recorded on a smoked paper on a kymograph by means of a frontal writing lever. The agonistic substances were added to the organ bath in a sub-maximal dosage, remaining in contact with the muscle for 30-60 sec and then washed out. Antagonists and toxin were allowed to act for periods of up to 5 min . Drugs used : acetylcholine chloride (Roche), adrenaline hydrochloride (Parke-Davis), atropine sulphate (Sigma), cocaine hydrochloride (Merck), physostigmine (eserine) sulphate (Merck), pentolinium tartrate (Ansolysen,® Wyeth), hexamethonium chloride (Squibb), methysergide maleate (Sandoz), phentolamine mesylate (Ciba), propranolol (Inderal,® Ayerst), 5-hydroxytryptamine creatinine sulphate (serotonin, Koch-Light), nicotine (Sigma), 'This paper was supported by Conselho National de Pesquisas and Conselho de Pesquisas da UFMG., Brazil. tFellows of CNPq., Brazil. 81
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bradykinin (Sandoz, BRS-640, Batch no. 69055) and substance P.* Tityus serrulatus venom was obtained from Instituto Butantan (Sâo Paulo, Brazil) and purified toxin fraction (T~ was prepared usinga combination of precipitation and column chromatography on sephadex G-25 (GOMFZ and DtNIZ, 1966) . Samples of purified toxin, kindly provided by Drs. M. V. Gomez and C. R. Diniz, from the Dept. of Biochemistry, 1CB, Belo Horizonte, Brazil, are devoid of phospholipase A and other enzymatic activities . The quantities of toxin are expressed as weights ofprotein . RESULTS
E ~ect of toxin on the rat ileum. When toxin (1-5 tag/ml) was added to the organ bath containing rat ileum, the muscle contracted 3-5 cm. In this preparation acetylcholine (5~5 x 10-8 to 1 ~1 x 10-'M), bradykinin (4~7 x 10 - e to 9-4 x 10-°M) and 5-HT (6~4 x 10-' to 2~6 x 10-°M) produced equiactive contractions (20 exp). The contraction induced by toxin is of a slow type, starting after a latent interval ranging from 10 to 60 sec, thereby differing from that produced by acetylcholine, bradykinin and 5-HT (Fig. 1 A). Following the slow response the muscle showed spasmodic contractures which lasted for 2 to 4 hr, even after repeated washings with Jalon solution . However, addition of atropine to the organ bath (1 ~5 x 10-'M) brought the muscle back to its original tone. In 10 out of 20 experiments a reinforcement of pendular movements and appearance of rhythmic contractions were also observed (Fig. 1A). Subsequent doses of toxin produced responses of smaller magnitude or no response at all (tachyphylaxis). Fig. 1B shows one of these experiments . On the other hand, larger doses of toxin (10 tcg/ml) produced fast contractions which returned to the base line 5-10 min after washing out the preparation (20 exp). In 10 experiments a relaxation was observed after the second addition of toxin ; the latter effect is not tachyphylactic, since it can be repeated by subsequent additions of the same doses of toxin . Mode ofaction oftoxin on the rat ileum. In order to determine the possible participation of acetylcholine in the mechanism of toxin action the rat ileum was suspended in a Jalon solution containing atropine (1 ~5 x 10-'M) . Under these circumstances, the addition of toxin to the bath (5-10 Fig/ml) produced in 8 out of 16 experiments a small relaxation which preceded the contraction (Fig. 2A). The reinforcement of pendular movement and rhythmic contractions were also recorded after addition of toxin to atropinized preparations. On the other hand, the addition of eserine to the bath (1 ~5 x 10-~M) potentiated the contraction produced by toxin (1 t`g/ml) ; moreover, subliminar doses of toxin (0~1 ~g/ml) produced contraction of the eserine treated preparations . Eserine was able to increase the muscular spasm produced by a previous dose of toxin (1 S exp). Two ofthese experiments are shown in Fig. 2, B and C. Ganglionic blockade with pentolinium (1 ~8 x 10-6M) or hexamethonium (3 ~7 x I O-°M) failed to prevent contractions produced by toxin (1-5 tag/ml, 5 exp). These results agree with those reported by DtrIIZ and GorrçALVES (1956) in the guinea-pig ileum. Serotonin (2~6 x 10-' to 2~6 x 10-°M) when added to an ileum segment which had previously received toxin (1 to 5 tag/ml), either failed to contract the preparation or evoked contractions smaller than those obtained before toxin (16 exp). Methysergide (1 ~1 x 10-°M) blocked the effects of Serotonin on the ileum, but had no influence on the action of toxin (7 exp). Atropine and methysergide when added simultaneously to the bath abolished the effects of acetylcholine "Kindly provided by Prof. F. Lembeek, from the Institute of Pharmacology, University of Graz, Universitätsplatz 4, A-ß0I0 Graz, Austria. TOXICON J973 YoJ. JJ .
FIG. 1 . RAT ILEUM PREPARATION SUSPENDED IN 1O ml JALON SOLUTION . Acetylcholine (ACh). 1 ~1 x 10 'M ; bradykinin (Bk), 9~4 r 10 -°M ; serotonin (5-HT), 6~4 x l0-'M and purified scorpion toxin (T), 3 ~g/ml. After each addition to the bath the preparation was rinsed once or twice ( 1 1 ). In tracing B, a period of 30 min elapsed between thefirstand the second addition of toxin ('T) .
FIG. 2. RAT ILEUM PREPARATIONS SUSPENDED IN IO ml JALON SOLUTION .
Tracing A. Acetylcholine (ACh), 2~7 :~ 10 -~M ; bradykinin (Bk), 1~9 : : 10-°M and purified scorpion toxin (T), 10 Fig/ml ; atropine (1~5 " 10-'M) was present in the bath throughout . Tracing B. Acetylcholine (ACh), 1~1 ~ 10 -"M ; toxin (T), 0~1 l+g/ml and eserine (E), I~5 ï": 10-'M . Tracing C. Toxin (T), 1 Fig/ml ; acetylcholine and eserine, as in B. After each addition to the bath the preparation was rinsed once or twice ( 1 1 ). roxfcoN ~ms v~r. m.
f.p . 82
FIG. 3. RAT ILEUM PREPARATIONS SUSPENDED IN lO mI JALON SOLUTION . Tracing A. Acetylcholine (ACh), 1~1 ": 10- 'M ; serotonin (5-HT), 7-8 >: 10 - ~M and purified scorpion toxin ('~, 2 pg/ml. A period of 30 min elapsed between the additions of toxin and 5-HT. Tracing B. Acetylcholine (ACh), 1 ~1 a ]0-'M ; serotonin (5-HT), 2~6 x 10-'M and purified scorpion toxin (T), 5 ~g/ml. At larger arrow, atropine (1 ~S x 10 -'M) and methysergide (1 ~1 >~ 10-"M) were added to the bath . ! 1 represents rinsing of the preparation .
TOXICON 1973 Vol. 11 .
FIG. 4. RAT ILEUM PREPARATION SUSPENDED IN IO ml .TALON SOLUTION . Adrenaline (Ad), 2~3 x 10 -'M ; nicotine (Nic), I~5 x 10-"M ; acetylcholine (ACh), 5~5 f 10 -°M and purified scorpion toxin (T), 10 l+g/ml . After each addition, the preparation was rinsed once or twice ( 1 1 ). A period of 15 min elapsed between the first dose of toxin and the second dose of adrenaline. In tracing B, phentolamine (2~6 x 10-°M) and propranolol (1 ~7 :< 10-"M) were maintained in the bath throughout the experiments.
FIG. S . RAT DUODENUM PREPARATIONS SUSPENDED IN IO ml JALON 30LUTION, Tracings A and B, atropine (I ~5 :~: 10- 'M) was maintained in the bath throughout the expts Adrenaline (Ad), 2~3 x 10- 'M ; bradykinin (Bk), 9~4 :~: ]0 -°M ; acetylcholine (ACh), 5 ~5 ~ 10-'M and purified scorpion toxin (T), 21~g/ml. In tracing B, phentolamine (2~6 ~ 10-°M) and propranolol (1 ~7 :< 10-°M) were present in the bath throughout the expts. At arrows, the bath fluid was changed. TOXICON 1973 Vol. 11 .
Swrpion Tozin on Rat Intestine
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and serotonin but did not prevent the contraction produced by toxin (6 exp) . Two of these experiments are shown in Fig. 3, A and B. Cocaine (7~4 x 10 -6 M) prevented the contraction produced by toxin (1-5 Fig/ml), but had no effect on the response caused by acetylcholine (5~5 x l0-e to 1 ~1 x 10-'M, 6 exp) . These results also agree with those described by Dirnz and Goxçnr vi:.s (1956) in the guinea-pig ileum. Propranolol (1 ~7 x 10-6 M) and phentolamine (2.6 x 10-bIV17 prevented the relaxation produced by toxin (10 hg/ml) and adrenaline (2 ~3 x 10-'M, 6 exp) . The results of one of these experiments are shownin Fig. 4. Addition of nicotine to the bath (6~2 x 10-8 to 1 ~5 x 10 -bM) caused contraction of the ileum. After toxin, the same dose of nicotine produced relaxation (20 exp), which was prevented either by propranolol and phentolamine (Fig . 4) or pentolinium (1 ~8 x 10 -°M). Reinforcement of pendular movements and rhythmic contractions caused by toxin were similar to those produced by substance P (0~5 mg/ml) . E ~ect of toxin on the rat duodenum . Addition of toxin to the rat duodenum (1-5 hg/ml) suspended in atropinized Jalon solution produced relaxation of the intestine (20 exp) . This effect was abolished by propranolol and phentolamine added simultaneously to the bath, before toxin (Fig . ~. As in the ileum, toxin caused contraction and increased the amplitude of pendular movements of the non-atropinized duodenum (8 exp) . DISCUSSION Isolated smooth muscle contractions caused by scorpion venom (Tityus serrulatus) have been claimed to be due to the release of an acetylcholine-like substance (Dixlz and GONÇALVFS, 1956 ; Dmnz and Vnr mi, 1959 ; Dnviz and ToxRES, 1968). In the present experiments, however, it was shown that rat ileum contractions produced by purified scorpion toxin were only partially blocked by atropine. As the contractions were not blocked by methysergide, the participation of 5-HT on the mechanism of contraction produced by toxin can be ruled out. The enhancement of intestinal tonus could be due to a continuous release of a mediator or mediators which cause a prolonged state of contraction . One of these mediators probably is acetylcholine, since eserine increased and atropine decreased the muscular spasm. In addition, the reinforcement of pendular movement and rhythmic variations of tonus caused by toxin, even in atropinized preparations, could be due to release of substance P. This hypothesis is supported by experiments showing reduction in the substance P content of rat gut after injection of scorpion toxin (HIAL and Drnnz, 1971). The tachyphylaxis observed after toxin could be due to inability of the ileum to release active substance (s), for the actions of acetylcholine and substance P were not abolished. On the other hand, the reduced effect shown by serotonin after toxin could be ascribed to the blockade of M receptors (GADDUM and PICARELLI, 1957) ; however, the number of these receptors in rat gut seems to be very small (LEVY and Mtet~,-B~t, 1956 ; GARATI'INI and VALZELLI, 196 . As substance P is able to block the contraction produced by serotonin (BELESLIN and VAR.AGIC, 1960), it is suggested that inhibition of the effect of 5-HT is related to the release of substance P from the intestine by scorpion toxin. Another possibility is that toxin blocks D receptors. The relaxation induced by toxin depends on an adrenergic mechanism, since alpha and beta sympatholytic drugs abolished it. These experiments are in accordance with a previous paper from this laboratory, which shows that the hypertensive effect of toxin in anesthetized rats is due to release of catecholamines (FytEIRE-MAm and DINIZ, 1970). The inversion of the 710XICON 1973 Yol. ll . E
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J. R. CUNHA MELO, L. FREIRE-MAIA, W. L. TAFURI and T. A. MARIA
nicotine effect produced by scorpion toxin ~in the rat ileum is similar to that described by AMSncxe (1951) in the rabbit ileum treated with botulinum toxin . It seems that scorpion toxin blocks the release of acetylcholine caused by nicotine . In addition, since the relaxation caused by nicotine is abolished both by ganglionic blockade and sympatholytic drugs it seems likely that it is due to release of catecholamines . Scorpion toxin causes a decrease in the number of the granular vesicles and a simultaneous increase in the agranular vesicles in the myenteric plexus of the rat (TAFIJRI et al., 1971) . It seems likely that the reduction in the number of the granular vesicles could be related to the release ofcatecholamines and perhaps substance P. Acknowledgement-The Authors express their sincere appreciation to Prof. W. T. BERALDO for helpful criticism of the manuscript and Mr . José Lutz Dw Cosrw for technical assistance . REFERENCES A~wcEtt:, N. (1951) Unmasking, after cholinergic paralysis by botulinum toxin, of a reversed action of nicotine on the mammalian intestine, revealing the probable presence of local inhibitory ganglion cells in the enteric plexuses. Br. J. Pharmac. 6, Sl . BELe4LIN, D. and Vwxwctc, V. (1960) The effort of substance P on the responses of the isolated guinea-pig ileum to acetylcholine, nicotine, histamine and 5-hydroxytryptamine. Archs int. Pharmacodyn. Thér. 126, 321 . Battra, J. H., Ftxrvt:v, D.J. and Goonwnv, L. G. (1950) Biological standardization, Sec . edition, p. 223 . London Oxford University Press. CtJxttw Mta.O, J. R, FRF14F-Mwu, L., TAFUttt, W. L. and Mwnu, T. A. (1970) Mecanismo de açito da toxins purificada de esoorpillo sobre o 11eo isolado de rato . Acrd physiol. latinoam . 20, 296. DEt, Pozo, E. D. (1956) Mechanism of pharmacological actions of scorpion venoms . In : Venoms (BUCKLEY, E. E. and POxaFS, N., Eds.), p. 123. Washington : Am . Ass. Adv. Sri. Dttatz, C. R. and Gotaçwt.ves, J. M. (1956) Some chemical and pharmacological properties of Brazilian scorpion venoms. In : Venoms (BuctcLEV, E. E. and Pondes, N., Eds.), p. 131 . Washington : Am . Ass. Adv. Sci. Dtraz, C. R. and Vwt.sw, V. (1959) Effects of a toxin present in a purified extract of telsons from thescorpion Titvus serrulatus on smooth muscle preparations and in mice. Archs inr. Pharmacodyn . Thér. 121, 1 . Dnvtz, C. R. and Totuees, J. M. (1968) Release of an acetylcholine-like substance from guinea-pig ileum by scorpion venom. Toxlcon 5, 277. Ftt~-Mwu, L. and Dttatz, C. R. (1970) Pharmacological action of a purified scorpion toxin in the rat. Toxlcon 8, 132. GADDUM, J. H. and Ptcwxia.u, Z. P. (1957) Two kinds of tryptamine receptor . Br . J. Pharmac. 12, 323. Gwrcwrnru, S. and Vwtzaru, L. (1965) Seroronin, p. 87 . Amsterdam: Elsevier . Go~z, M . V. and Dtrrtz, C. R. (1966) Separation of toxic components from the Brazilian scorpion-Tiryus serrulatus-venom. Mrnrs Isst . Butantan 33, 899. Hut., W. and Dnatz, C. R. (1971) Efeito da scorpiotoxina sobre o conteudo de substltncia P do intestino de rato . Ciénc. Cult ., S. Paulo, 23 (supl), 304. LEVY, J. and Mtcttet.-Brat, E. (1956) . Contribution a l'action pharmacologique exercée par la sdrotonine sur quelques organes isolés (intestin et oreillette). J. Physlol., Paris 48. 1051 . Pwrreagorr, R. A. (1962) Pharmacologie action of scorpion venom on intestinal smooth muscle. Toxic. rrppl. Pharmac. 4, 710. Tw~ttt, W. L., Mwww, T. A., FREIRE-MAIA, L. and C~trattw MEt.o, J. R. (1971) Effect of purified scorpion toxin on vesicular components in the myenteric plexus of the rat. Toxlcon 9, 427.
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