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Effect of scorpion toxin from tityus serrulatus on the contraction of the isolated rat uterus
Foxicon.Vol. 33, No. 3, pp.
Pergamon
EFFECT ON THE
355-361. 1995 Copyfisht ~'3 1995 Elsevier Scknc¢ Ltd Printed in Great Britain. All rishts res~trved 0041-0101/95 $9.50 + 0.(30
0041-0101(94)00162-6
OF SCORPION
TOXIN
CONTRACTION
FROM
TITYUS
OF THE ISOLATED
SERRULATUS RAT UTERUS
M. MENDON(~.A, 1 M. M. PROFETA DA LUZ, 2 L. FREIRE-MAIA 3 and J. R. CUNHA-MELO 2. IDepartamento de Ginecologia,2Departamentode Cirurgia da Faeuldadede Medicina, e 3Departamentode Farmaeologiado Institutode Ci~nciasBiol6gieas,UniversidadeFederalde MinasCrerais,BeloHorizonte,Brazil (Received
17
June
1994;
accepted
28
September
1994)
M. Mendonga, M. M. Profeta Da Luz, L. Freire-Maia and J. R. Cunha-Melo. Effect of scorpion toxin from Tityus serrulatus on the contraction of the isolated rat uterus. Toxicon 33, 355-361, 1995.--Scorpion toxin TI from Tityus serrulatus was tested for its effects on the isolated rat uterus preparation. T I (5/~g/ml) caused a contraction of the uterus, which was potentiated by neosligrnine (!.64 x 10 -6 M) and abolished by atropine (i.4 x 10 -7 M ) . After addition of neostigmine to the bath, we noted a higher amplitude of the toxininduced contractions, and the appearance of repetitive rhythmic contractions. The :~corpion toxin-induced contraction was not prevented by previous addition to the bath of hexamethonium or bradykinin, 5-HT and angiotensin II antagonists. The uterine contraction was prevented by previous addition to the bath of either tetrodotoxin (5 x 10 -8 M) or lidocaine (4.2 x 10 -5 M). These data seem to indicate that scorpion toxin-induced rat uterus contractions are due to actions on post-ganglionic autonomic nerve endings, with acetylcholine release and stimulation of muscarinic receptors.
INTRODUCTION Although the innervation of mammalian uterus has been studied extensively, precise information regarding the intrinsic autonomic neural pathways and the relationship between the cholinergic and adrenergic components is lacking. The classical histologic methods employed for demonstration of nerve fibers are non-specific, allowing only a differentiation between myelinated and nonmyelinated nerves. This has provided conflicting interpretations of data regarding the distribution of intrinsic ganglia and ganglionic cells (Adham and Schenk, 1969). The myometrium innervation and the participation of neurogenic control of uterine contractility have not been clearly defined. It is generally accepted that neurogenic mechanisms play no role in the control of myometrial contractility. The uterus of a number of species is thought to be primarily innervated by post-ganglionic adrenergic fibers from the sympathetic nervous system. Cholinergic innervation would be sparse and mainly related to parametrial blood vessels (Garfield, 1986). *Author to whomcorrespondenceshould be addressed. "~
355
356
M. MENDON(~A et al.
The rich innervation observed in the urinary bladder was not found in the myometrium. It is possible that the presence of a large number of cellular contacts between myometrial smooth muscle cells facilitates the propagation of action potentials from one muscle cell to another and thus obviates the necessity for rich innervation (Silva, 1966). Much work has been devoted to uterine sympathetic nerves with no corresponding interest concerning the cholinergic nerves, despite evidence for cholinergic innervation in the uterus of different mammalian species, particularly rat (Adham and Schenk, 1969) and human. The cholinergic fibers in the human cervix show an overall distribution similar to that of adrenergic fibers, i.e. they are rare in the uterine corpus and numerous in the cervix and isthmus (Stjernquist and Owman, 1985). On the one hand, some investigators have reported that uterine effects evoked by Leiurus quinquestriatus, Pandinus exitialis and Androctonus amoureuxi scorpion venoms are due to the release of kinins, prostaglandins and/or slow-reacting substances (Osman et al., 1972; Ismail et al., 1974, 1983). On the other hand, neurotoxins from the Brazilian scorpion Tityus serrulatus are toxins that act on the post-ganglionic nerve endings, interacting with the sodium channels, with subsequent release of chemical mediators such as acetylcholine and catecholamines (Freire-Maia and Campos, 1989). They represent a good tool for studies on the mechanisms of neural control in organs innervated by the autonomic nervous system (Catterall, 1975). The objective of this paper was to study the effects of T. serrulatus scorpion toxin on isolated rat uterus in an attempt to clarify some aspects of the neural control of this organ. A preliminary report of this study was published elsewhere (Costa Cruz et al., 1973). MATERIALS AND METHODS Seventy-four albino, adult virgin female Holtzman rats weighing 100-200 g were injected i.m. with 0.5 mg estradiol benzoate 72 hr before the experiments. The animals were sacrificed by cervical dislocation, and exsanguinated by cutting the cervical vessels. The abdomen was opened and the uterine horns were removed, freed of fat and immersed in a modified Tyrode solution with the following composition in mmole/litre: NaCI 136.8; KC1 2.7; MgCI 2 2.1; CaC12 0.72; NaH2PO 4 0.4; NaHCO 3 11.9; glucose 5.5 (pH 7.4). One uterine horn segment measuring 3 cm was suspended in a 5 ml smooth muscle bath containing modified Tyrode solution at 34°C, bubbled with atmospheric air and connected to a Grass transducer. The isometric recording of uterine musculature activity was done with a FTO3 transducer connected to a Grass polygraph model 7D (Grass, Quincy, MA, U.S.A.). The transducer was previously calibrated to establish a relationship between the force applied to the transducer and the gauge deflection (1 g = 20 mm). The preparations were allowed to equilibrate in the bath for 30 min before the experiments. Scorpion toxin, agonists and antagonists were directly added to the reservoir containing the uterus. The preparation was washed twice with the modified Tyrode solution after addition of the agonists or toxin and allowed to rest for 3 min before the next addition. Toxin effects were compared with those of the other substances utilized and the results were analyzed based on the amplitude of contractions in the absence and presence of antagonists. Scorpion toxin and drugs Scorpion toxin--purified T~ fraction from the Brazilian scorpion Tityus serrulatus (Gomez and Diniz, 1966)--was kindly supplied by Professor Tasso Moraes-Santos (Faculdade de Farmficia, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil); acetylcholine chloride (F.W. 181.7), 5-hydroxytryptamine, 5-HT (F.W. 387.4), atropine sulfate (F.W. 676.8), nicotine alkaloid (95-98%), tetrodotoxin (F.W. 319.3) and hexamethonium bromide (F.W, 362.21) were from Sigma (St Louis, MO, U.S.A.); bradykinin (F.W. 1060.2), oxytocin (F.W. 1007.23) and methysergide (F.W. 353.45) were from Sandoz (Santo Amaro, S.P. Brazil); angiotensin II (F.W. 1046.2) were synthesized by Dr A. Paiva and Dr L. Juliano (Escola Pualista de Medicina, S.P., Brazil); HOE 140: D-Arg[Hyp-3-di-5-o-Tic-7-Oc-8]bradykinin (F.W. 1304.59) was from Hoechst (Suzano, S.P. Brazil); DUP753: [2-n-buty•-4-ch••r•-5-hydr•ximethy•-I[[2'-(•H-tetraz••-5-y•)bipheny•-4-y•]]Methy•imidaz••e (F.W. 500.0) was from Dupont (Boston, MA., U.S.A.); neostigmine bromide (F.W. 303.2) was from Roche (Rio de Janeiro, R.J. Brazil) and lidocaine hydrochloride (F.W. 270.8) was from Cristfilia (Itapira, S.P. Brazil).
Contraction of Rat Uterus Induced by Scorpion Toxin
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Fig. 1. Effect of scorpion toxin and acetylcholine on rat uterus. The uterus was suspended in 5 ml modified Tyrode solution at 34°C. Ach, Acetylcholine (1.10 x 10-7 M); Tox, scorpion toxin (fraction T~, 5.0 #g/ml). The preparation was washed at the platea~L of contraction (arrows). Interval between doses = 4 rain. A: Observe isolated contractions following toxin addition; B: repeated contractions following toxin addition
RESULTS
Uterine response to scorpion toxin (fraction Tl) Addition of 5/tg/ml of fraction T~ to the bath caused contraction in 8 out of 13 experiments, after a latency of 10-40 sec. The contractions were usually isolated. In these preparations, acetylcholine at a dose of 1.1 x 10 -7 M caused contractions with a mean amplitude of 33.2 mm in height, corresponding to a force of 2.32 g (Fig. 1A). In some cases, when scorpion toxin was allowed to act for several minutes before washing of the preparation, repeated contractions were observed at variable intervals (Fig. 1B).
Effects of drugs on the contractions caused by scorpion toxin (fraction T~) Neostigmine and atropine. Neostigmine (1.64 x 10 ~6 M ) potentiated the effect of fraction T~. When toxin was added to unwashed neostigmine-treated uterus, several contractions followed by relaxations were observed. This pattern lasted for several min and was
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Fig. 2. Effeci of neostigmine and atropine on the scorpion toxin-induced contraction of the rat uterus. The toxin (fraction Ti) induced a sustained contraction, with superimposed contractions and incomplete relaxations, when added to a modified Tyrode solution containing neostigmine. The contractions were abolished by atropine. The experimental conditions were similar to those described in Fig. 1. Ach, Acetylcholine (l.10 x 10-7 M); Tox, scorpion toxin (5#g/ml); Ne, neostigmine (1.64 x 10-6M); At, atropine (1.4 x 10-7 M); BK, bradykinin (3.8 x 10-~°M). The arrows indicate washing of the preparation.
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Contraction of Rat Uterus Induced by ScorpionToxin
359
interrupted by atropine (Fig. 2). Addition of neostigmine alone to the bath did not evoke any response. The previous addition of atropine (1.4 x 10-7 M) to the bath prevented the contractions induced by aeetylcholine (1.1 x 10-7 M ) o r fraction T ~ - 5 #g/ml (data not shown).
Lidocaine. Previous addition of 4.2 x 10-SM lidocaine hydrochloride to the bath prevented the contraction caused by 5/zg/ml fraction Tt. After repeated washings, a new addition of fraction T~ 30 min after the previous dose induced contractions similar to those observed in the lidocaine-free preparations. Tetrodotoxin (TTX). The addition of fraction T~ (5 #g/ml) to the bath after 1.64 x 10-6 M neostigmine caused repeated contractions of the uterus. This effect was abolished by 5 x 10-~M TTX. After washing, the uterus started to contract again with the same pattern as observed in TTX-free preparations, even if no additional dose of toxin was added. A new addition of TTX to the bath abolished the contractions induced by fraction Tt (Fig. 3). Bradykinin blocker (HOE140). The addition of 7.6 x 10 -s M HOE 140 to the bath was able to prevent the contraction induced by 3.8 x 10-~0 M bradykinin, but had no effect on the contraction induced by 5 #g/ml of fraction T~. 5-Hydroxytryptamine (5-HT) blocker (methysergide--MTS). The contractions caused by the addition of 2 x 10-SM 5-HT were prevented when 1.13 x 10-SM of the 5-HT blocker MTS was added to the bath. This dose of MTS did not prevent the contraction caused by 5 #g/ml fraction T~. Angiotensin H blocker (DUP753). The addition of 1.60 x 10 -6 M DUP753 to the bath prevented tile contractions caused by 4.0 x 10 -9 M angiotensin II. The contractions caused by 5 #g/ml fraction T~ were unaffected by the same dose of DUP753. Table 1 summarizes the effects of several drugs on the uterine contractions induced by fraction T~. Nicotine. Nicotine at a dose of 1 x 10-6 M to 1 x 10 -4 M did not cause contractions of the uterus (8 experiments). Hexamethonium. The ganglionic blocker hexamethonium at a dose of 1.9 x 10 -6 M did not prevent the contractions induced by 5#g/ml fraction Tl or 1.10x 10-TM acetylcholine. DISCUSSION It is well known that scorpion toxin exerts its effects by stimulation of the nerve endings of the autortomic nervous system (Freire-Maia and Campos, 1989). However, the possible effect of scorpion toxin on rat uterus has not been investigated. In the present experiments TON 33/3~E
360
M. MENDON(~A et al.
we have shown that the toxin induced isolated contraction of the preparation and, in some cases, a single dose of toxin evoked repeated contractions. Additions of specific antagonists of 5-HT, bradykinin and angiotensin II to the bath prevented the contractions induced by these mediators but did not prevent the effect of the toxin. Therefore, the data indicate that the uterus contraction elicited by scorpion toxin is not related to the release of these mediators. As the effects of toxin are potentiated by neostigmine and prevented or abolished by atropine, it seems likely that the action of toxin depends on stimulation of cholinergic muscarinic receptors. Since scorpion toxin acts on sodium channels (Gomez et al., 1973; Freire-Maia et al., 1975, 1976; Catterall, 1975) we decided to block these channels with tetrodotoxin (TTX). Our data showed that TTX did not prevent the contraction induced by acetylcholine or bradykinin but totally abolished the contraction elicited by fraction Tj (Table 1, Fig. 3). After washing the preparation, the uterus started to contract, even if no additional dose of fraction Tl was added (Fig. 3). This could be explained assuming that TTX was removed by washing, whereas the fraction Tl remained bound to the sodium channels, releasing acetylcholine, as was suggested in other preparations by Freire-Maia et al. (1975, 1976) and Catterall (1975). Therefore, the experiments with TTX, neostigmine and atropine (Figs 1-3) indicate that the uterine contraction elicited by scorpion toxin is due to release of acetylcholine from post-ganglionic nerve fibers, with subsequent stimulation of muscarinic receptors. Garfield (1986), studying uterine musculature of non-pregnant rats, and using methods for acetylcholinesterase (AChase) distribution, showed that AChase was located around blood vessels as well as near myometrial cells. Nevertheless, identification of acetylcholinesterase-positive endings is considered indicative but not sufficient for the detection of cholinergic neurons. However, transmural stimulation of the rat uterus produced cervical and uterine horn contractions which were blocked by hyoscine or tetrodotoxin, suggesting a cholinergic motor innervation (Hollingsworth, 1974). If we assume that the contraction of rat uterus induced by scorpion toxin is due to release of acetylcholine from intrinsic parasympathetic nerve endings, this constitutes a strong indication that the rat uterus is innervated by the parasympathetic division of the autonomic nervous system.
Acknowledgements--The authors would like to thank Professor Tasso Moraes-Santos (School of Pharmacy,
Federal University of Minas Gerais, Belo Horizonte, Brazil) for kindly supplying scorpion toxin (fraction T~). This work was supported in part by funds from the Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico (CNPq) and FAPEMIG, Brazil. J.R.C.M. and L.F.M. are fellows of the CNPq.
REFERENCES Adham, N. and Schenk, E. A. (1969) Autonomicinnervation of the rat vagina, cervix and uterus and its cyclic variation. Am. J. Obstet. Gynec. 15, 508 516. Catterall, W. A. (1975) Cooperative activation of action potential Na + ionophore by neurotoxins. Proc. natn. Acad. Sci. U.S.A 72, 1782-1786. Costa Cruz, T. A., Freire-Maia, L. and Beraldo, W. T. (1973) A~:fioocit6cica da tityustoxina. Cienc. Cult. (S~o Paulo) 25 (suppl.), 283. Freire-Maia, L. and Campos, J. A. (1989) Pathophysiologyand treatment of scorpion poisoning. In: Natural Toxins--Characterization, Pharmacology and Treatment, pp. 139 159 (Ownby, C. L. and Odell, G. V., Eds). Oxford: Pergamon Press. Freire-Maia, L., Cunha-Melo, J. R., Gomez, M. V., Tafuri, W. L., Maria, T. A. and Futuro-Neto, H. A. (1975) Studies on the mechanism of action of tityustoxin. Toxicon 13, 93.
Contraction of Rat Uterus Induced by Scorpion Toxin
361
Freire-Maia, L., Cunha-Melo, J. R., Gomez, M. V., Tafuri, W. L., Maria, T. A., Calixto, S. L. and Futuro-Neto, H. A. (1976) Studies on the mechanism of action of tityustoxin. In: Animal, Plant and Microbial Toxins, Vol. 2, pp. 273-285 (Ohsaka, A., Hayashi, K. and Sawai, Y., Eds). New York: Plenum Press. Garfield, R. E. (1986) Structural studies ofinnervation on non-pregnant rat uterus. Am. J. Physiol. 251, C41-C54. Gomez, M. V. and Diniz, C. R. (1966) Separation of toxic components from the Brazilian scorpion--Tityus serrulatus----~renom. Mere. Inst. Butantan 33, 899-902. Gomez, M. V., Dai, M. E. and Diniz, C. R. (1973) Effect of scorpion venom, tityustoxin, on the release of acetylcholine from incubated slices of rat brain. J. Neurochem. 20, 1051-1061. Hollingsworth, M. (1974) The innervation of the rat cervix and its pharmacology in vitro and in vivo. Br. J. Pharmac. 52, 539-547. Ismail, M., Osman, O. H., Gumaa, K. A. and Karrar, M. A. (1974) Some pharmacological studies with scorpion (Pandinus exitialis) venom. Toxicon 12, 75-82. Ismail, M., Ellison, A. C. and Tilmisany, A. K. (1983) Teratogenicity in the rat of the venom from the scorpion Androctonus amoureuxi (Aud. &Sav.). Toxicon 21, 177-189. Osman, O. H., Ismail, M., El-Asmar, M. F. and Ibrahim, S. A. (1972) Effect on the rat uterus of the venom from the scc,rpion Leiurus quinquestriatus. Toxicon 10, 363-366. Silva, D. G. (1966) The ultrastructure of myometrium of the rat with special reference to the innervation. Anat. Rec. 158, 21-34. Stjernquist, M. and Owman, C. (1985) Cholinergic and adrenergic neural control of smooth muscle function in the non-pre[~ant rat uterine cervix. Acta Physiol. scand. 124, 429-436.
Pergamon
EFFECT ON THE
355-361. 1995 Copyfisht ~'3 1995 Elsevier Scknc¢ Ltd Printed in Great Britain. All rishts res~trved 0041-0101/95 $9.50 + 0.(30
0041-0101(94)00162-6
OF SCORPION
TOXIN
CONTRACTION
FROM
TITYUS
OF THE ISOLATED
SERRULATUS RAT UTERUS
M. MENDON(~.A, 1 M. M. PROFETA DA LUZ, 2 L. FREIRE-MAIA 3 and J. R. CUNHA-MELO 2. IDepartamento de Ginecologia,2Departamentode Cirurgia da Faeuldadede Medicina, e 3Departamentode Farmaeologiado Institutode Ci~nciasBiol6gieas,UniversidadeFederalde MinasCrerais,BeloHorizonte,Brazil (Received
17
June
1994;
accepted
28
September
1994)
M. Mendonga, M. M. Profeta Da Luz, L. Freire-Maia and J. R. Cunha-Melo. Effect of scorpion toxin from Tityus serrulatus on the contraction of the isolated rat uterus. Toxicon 33, 355-361, 1995.--Scorpion toxin TI from Tityus serrulatus was tested for its effects on the isolated rat uterus preparation. T I (5/~g/ml) caused a contraction of the uterus, which was potentiated by neosligrnine (!.64 x 10 -6 M) and abolished by atropine (i.4 x 10 -7 M ) . After addition of neostigmine to the bath, we noted a higher amplitude of the toxininduced contractions, and the appearance of repetitive rhythmic contractions. The :~corpion toxin-induced contraction was not prevented by previous addition to the bath of hexamethonium or bradykinin, 5-HT and angiotensin II antagonists. The uterine contraction was prevented by previous addition to the bath of either tetrodotoxin (5 x 10 -8 M) or lidocaine (4.2 x 10 -5 M). These data seem to indicate that scorpion toxin-induced rat uterus contractions are due to actions on post-ganglionic autonomic nerve endings, with acetylcholine release and stimulation of muscarinic receptors.
INTRODUCTION Although the innervation of mammalian uterus has been studied extensively, precise information regarding the intrinsic autonomic neural pathways and the relationship between the cholinergic and adrenergic components is lacking. The classical histologic methods employed for demonstration of nerve fibers are non-specific, allowing only a differentiation between myelinated and nonmyelinated nerves. This has provided conflicting interpretations of data regarding the distribution of intrinsic ganglia and ganglionic cells (Adham and Schenk, 1969). The myometrium innervation and the participation of neurogenic control of uterine contractility have not been clearly defined. It is generally accepted that neurogenic mechanisms play no role in the control of myometrial contractility. The uterus of a number of species is thought to be primarily innervated by post-ganglionic adrenergic fibers from the sympathetic nervous system. Cholinergic innervation would be sparse and mainly related to parametrial blood vessels (Garfield, 1986). *Author to whomcorrespondenceshould be addressed. "~
355
356
M. MENDON(~A et al.
The rich innervation observed in the urinary bladder was not found in the myometrium. It is possible that the presence of a large number of cellular contacts between myometrial smooth muscle cells facilitates the propagation of action potentials from one muscle cell to another and thus obviates the necessity for rich innervation (Silva, 1966). Much work has been devoted to uterine sympathetic nerves with no corresponding interest concerning the cholinergic nerves, despite evidence for cholinergic innervation in the uterus of different mammalian species, particularly rat (Adham and Schenk, 1969) and human. The cholinergic fibers in the human cervix show an overall distribution similar to that of adrenergic fibers, i.e. they are rare in the uterine corpus and numerous in the cervix and isthmus (Stjernquist and Owman, 1985). On the one hand, some investigators have reported that uterine effects evoked by Leiurus quinquestriatus, Pandinus exitialis and Androctonus amoureuxi scorpion venoms are due to the release of kinins, prostaglandins and/or slow-reacting substances (Osman et al., 1972; Ismail et al., 1974, 1983). On the other hand, neurotoxins from the Brazilian scorpion Tityus serrulatus are toxins that act on the post-ganglionic nerve endings, interacting with the sodium channels, with subsequent release of chemical mediators such as acetylcholine and catecholamines (Freire-Maia and Campos, 1989). They represent a good tool for studies on the mechanisms of neural control in organs innervated by the autonomic nervous system (Catterall, 1975). The objective of this paper was to study the effects of T. serrulatus scorpion toxin on isolated rat uterus in an attempt to clarify some aspects of the neural control of this organ. A preliminary report of this study was published elsewhere (Costa Cruz et al., 1973). MATERIALS AND METHODS Seventy-four albino, adult virgin female Holtzman rats weighing 100-200 g were injected i.m. with 0.5 mg estradiol benzoate 72 hr before the experiments. The animals were sacrificed by cervical dislocation, and exsanguinated by cutting the cervical vessels. The abdomen was opened and the uterine horns were removed, freed of fat and immersed in a modified Tyrode solution with the following composition in mmole/litre: NaCI 136.8; KC1 2.7; MgCI 2 2.1; CaC12 0.72; NaH2PO 4 0.4; NaHCO 3 11.9; glucose 5.5 (pH 7.4). One uterine horn segment measuring 3 cm was suspended in a 5 ml smooth muscle bath containing modified Tyrode solution at 34°C, bubbled with atmospheric air and connected to a Grass transducer. The isometric recording of uterine musculature activity was done with a FTO3 transducer connected to a Grass polygraph model 7D (Grass, Quincy, MA, U.S.A.). The transducer was previously calibrated to establish a relationship between the force applied to the transducer and the gauge deflection (1 g = 20 mm). The preparations were allowed to equilibrate in the bath for 30 min before the experiments. Scorpion toxin, agonists and antagonists were directly added to the reservoir containing the uterus. The preparation was washed twice with the modified Tyrode solution after addition of the agonists or toxin and allowed to rest for 3 min before the next addition. Toxin effects were compared with those of the other substances utilized and the results were analyzed based on the amplitude of contractions in the absence and presence of antagonists. Scorpion toxin and drugs Scorpion toxin--purified T~ fraction from the Brazilian scorpion Tityus serrulatus (Gomez and Diniz, 1966)--was kindly supplied by Professor Tasso Moraes-Santos (Faculdade de Farmficia, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil); acetylcholine chloride (F.W. 181.7), 5-hydroxytryptamine, 5-HT (F.W. 387.4), atropine sulfate (F.W. 676.8), nicotine alkaloid (95-98%), tetrodotoxin (F.W. 319.3) and hexamethonium bromide (F.W, 362.21) were from Sigma (St Louis, MO, U.S.A.); bradykinin (F.W. 1060.2), oxytocin (F.W. 1007.23) and methysergide (F.W. 353.45) were from Sandoz (Santo Amaro, S.P. Brazil); angiotensin II (F.W. 1046.2) were synthesized by Dr A. Paiva and Dr L. Juliano (Escola Pualista de Medicina, S.P., Brazil); HOE 140: D-Arg[Hyp-3-di-5-o-Tic-7-Oc-8]bradykinin (F.W. 1304.59) was from Hoechst (Suzano, S.P. Brazil); DUP753: [2-n-buty•-4-ch••r•-5-hydr•ximethy•-I[[2'-(•H-tetraz••-5-y•)bipheny•-4-y•]]Methy•imidaz••e (F.W. 500.0) was from Dupont (Boston, MA., U.S.A.); neostigmine bromide (F.W. 303.2) was from Roche (Rio de Janeiro, R.J. Brazil) and lidocaine hydrochloride (F.W. 270.8) was from Cristfilia (Itapira, S.P. Brazil).
Contraction of Rat Uterus Induced by Scorpion Toxin
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Fig. 1. Effect of scorpion toxin and acetylcholine on rat uterus. The uterus was suspended in 5 ml modified Tyrode solution at 34°C. Ach, Acetylcholine (1.10 x 10-7 M); Tox, scorpion toxin (fraction T~, 5.0 #g/ml). The preparation was washed at the platea~L of contraction (arrows). Interval between doses = 4 rain. A: Observe isolated contractions following toxin addition; B: repeated contractions following toxin addition
RESULTS
Uterine response to scorpion toxin (fraction Tl) Addition of 5/tg/ml of fraction T~ to the bath caused contraction in 8 out of 13 experiments, after a latency of 10-40 sec. The contractions were usually isolated. In these preparations, acetylcholine at a dose of 1.1 x 10 -7 M caused contractions with a mean amplitude of 33.2 mm in height, corresponding to a force of 2.32 g (Fig. 1A). In some cases, when scorpion toxin was allowed to act for several minutes before washing of the preparation, repeated contractions were observed at variable intervals (Fig. 1B).
Effects of drugs on the contractions caused by scorpion toxin (fraction T~) Neostigmine and atropine. Neostigmine (1.64 x 10 ~6 M ) potentiated the effect of fraction T~. When toxin was added to unwashed neostigmine-treated uterus, several contractions followed by relaxations were observed. This pattern lasted for several min and was
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Fig. 2. Effeci of neostigmine and atropine on the scorpion toxin-induced contraction of the rat uterus. The toxin (fraction Ti) induced a sustained contraction, with superimposed contractions and incomplete relaxations, when added to a modified Tyrode solution containing neostigmine. The contractions were abolished by atropine. The experimental conditions were similar to those described in Fig. 1. Ach, Acetylcholine (l.10 x 10-7 M); Tox, scorpion toxin (5#g/ml); Ne, neostigmine (1.64 x 10-6M); At, atropine (1.4 x 10-7 M); BK, bradykinin (3.8 x 10-~°M). The arrows indicate washing of the preparation.
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Contraction of Rat Uterus Induced by ScorpionToxin
359
interrupted by atropine (Fig. 2). Addition of neostigmine alone to the bath did not evoke any response. The previous addition of atropine (1.4 x 10-7 M) to the bath prevented the contractions induced by aeetylcholine (1.1 x 10-7 M ) o r fraction T ~ - 5 #g/ml (data not shown).
Lidocaine. Previous addition of 4.2 x 10-SM lidocaine hydrochloride to the bath prevented the contraction caused by 5/zg/ml fraction Tt. After repeated washings, a new addition of fraction T~ 30 min after the previous dose induced contractions similar to those observed in the lidocaine-free preparations. Tetrodotoxin (TTX). The addition of fraction T~ (5 #g/ml) to the bath after 1.64 x 10-6 M neostigmine caused repeated contractions of the uterus. This effect was abolished by 5 x 10-~M TTX. After washing, the uterus started to contract again with the same pattern as observed in TTX-free preparations, even if no additional dose of toxin was added. A new addition of TTX to the bath abolished the contractions induced by fraction Tt (Fig. 3). Bradykinin blocker (HOE140). The addition of 7.6 x 10 -s M HOE 140 to the bath was able to prevent the contraction induced by 3.8 x 10-~0 M bradykinin, but had no effect on the contraction induced by 5 #g/ml of fraction T~. 5-Hydroxytryptamine (5-HT) blocker (methysergide--MTS). The contractions caused by the addition of 2 x 10-SM 5-HT were prevented when 1.13 x 10-SM of the 5-HT blocker MTS was added to the bath. This dose of MTS did not prevent the contraction caused by 5 #g/ml fraction T~. Angiotensin H blocker (DUP753). The addition of 1.60 x 10 -6 M DUP753 to the bath prevented tile contractions caused by 4.0 x 10 -9 M angiotensin II. The contractions caused by 5 #g/ml fraction T~ were unaffected by the same dose of DUP753. Table 1 summarizes the effects of several drugs on the uterine contractions induced by fraction T~. Nicotine. Nicotine at a dose of 1 x 10-6 M to 1 x 10 -4 M did not cause contractions of the uterus (8 experiments). Hexamethonium. The ganglionic blocker hexamethonium at a dose of 1.9 x 10 -6 M did not prevent the contractions induced by 5#g/ml fraction Tl or 1.10x 10-TM acetylcholine. DISCUSSION It is well known that scorpion toxin exerts its effects by stimulation of the nerve endings of the autortomic nervous system (Freire-Maia and Campos, 1989). However, the possible effect of scorpion toxin on rat uterus has not been investigated. In the present experiments TON 33/3~E
360
M. MENDON(~A et al.
we have shown that the toxin induced isolated contraction of the preparation and, in some cases, a single dose of toxin evoked repeated contractions. Additions of specific antagonists of 5-HT, bradykinin and angiotensin II to the bath prevented the contractions induced by these mediators but did not prevent the effect of the toxin. Therefore, the data indicate that the uterus contraction elicited by scorpion toxin is not related to the release of these mediators. As the effects of toxin are potentiated by neostigmine and prevented or abolished by atropine, it seems likely that the action of toxin depends on stimulation of cholinergic muscarinic receptors. Since scorpion toxin acts on sodium channels (Gomez et al., 1973; Freire-Maia et al., 1975, 1976; Catterall, 1975) we decided to block these channels with tetrodotoxin (TTX). Our data showed that TTX did not prevent the contraction induced by acetylcholine or bradykinin but totally abolished the contraction elicited by fraction Tj (Table 1, Fig. 3). After washing the preparation, the uterus started to contract, even if no additional dose of fraction Tl was added (Fig. 3). This could be explained assuming that TTX was removed by washing, whereas the fraction Tl remained bound to the sodium channels, releasing acetylcholine, as was suggested in other preparations by Freire-Maia et al. (1975, 1976) and Catterall (1975). Therefore, the experiments with TTX, neostigmine and atropine (Figs 1-3) indicate that the uterine contraction elicited by scorpion toxin is due to release of acetylcholine from post-ganglionic nerve fibers, with subsequent stimulation of muscarinic receptors. Garfield (1986), studying uterine musculature of non-pregnant rats, and using methods for acetylcholinesterase (AChase) distribution, showed that AChase was located around blood vessels as well as near myometrial cells. Nevertheless, identification of acetylcholinesterase-positive endings is considered indicative but not sufficient for the detection of cholinergic neurons. However, transmural stimulation of the rat uterus produced cervical and uterine horn contractions which were blocked by hyoscine or tetrodotoxin, suggesting a cholinergic motor innervation (Hollingsworth, 1974). If we assume that the contraction of rat uterus induced by scorpion toxin is due to release of acetylcholine from intrinsic parasympathetic nerve endings, this constitutes a strong indication that the rat uterus is innervated by the parasympathetic division of the autonomic nervous system.
Acknowledgements--The authors would like to thank Professor Tasso Moraes-Santos (School of Pharmacy,
Federal University of Minas Gerais, Belo Horizonte, Brazil) for kindly supplying scorpion toxin (fraction T~). This work was supported in part by funds from the Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico (CNPq) and FAPEMIG, Brazil. J.R.C.M. and L.F.M. are fellows of the CNPq.
REFERENCES Adham, N. and Schenk, E. A. (1969) Autonomicinnervation of the rat vagina, cervix and uterus and its cyclic variation. Am. J. Obstet. Gynec. 15, 508 516. Catterall, W. A. (1975) Cooperative activation of action potential Na + ionophore by neurotoxins. Proc. natn. Acad. Sci. U.S.A 72, 1782-1786. Costa Cruz, T. A., Freire-Maia, L. and Beraldo, W. T. (1973) A~:fioocit6cica da tityustoxina. Cienc. Cult. (S~o Paulo) 25 (suppl.), 283. Freire-Maia, L. and Campos, J. A. (1989) Pathophysiologyand treatment of scorpion poisoning. In: Natural Toxins--Characterization, Pharmacology and Treatment, pp. 139 159 (Ownby, C. L. and Odell, G. V., Eds). Oxford: Pergamon Press. Freire-Maia, L., Cunha-Melo, J. R., Gomez, M. V., Tafuri, W. L., Maria, T. A. and Futuro-Neto, H. A. (1975) Studies on the mechanism of action of tityustoxin. Toxicon 13, 93.
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Freire-Maia, L., Cunha-Melo, J. R., Gomez, M. V., Tafuri, W. L., Maria, T. A., Calixto, S. L. and Futuro-Neto, H. A. (1976) Studies on the mechanism of action of tityustoxin. In: Animal, Plant and Microbial Toxins, Vol. 2, pp. 273-285 (Ohsaka, A., Hayashi, K. and Sawai, Y., Eds). New York: Plenum Press. Garfield, R. E. (1986) Structural studies ofinnervation on non-pregnant rat uterus. Am. J. Physiol. 251, C41-C54. Gomez, M. V. and Diniz, C. R. (1966) Separation of toxic components from the Brazilian scorpion--Tityus serrulatus----~renom. Mere. Inst. Butantan 33, 899-902. Gomez, M. V., Dai, M. E. and Diniz, C. R. (1973) Effect of scorpion venom, tityustoxin, on the release of acetylcholine from incubated slices of rat brain. J. Neurochem. 20, 1051-1061. Hollingsworth, M. (1974) The innervation of the rat cervix and its pharmacology in vitro and in vivo. Br. J. Pharmac. 52, 539-547. Ismail, M., Osman, O. H., Gumaa, K. A. and Karrar, M. A. (1974) Some pharmacological studies with scorpion (Pandinus exitialis) venom. Toxicon 12, 75-82. Ismail, M., Ellison, A. C. and Tilmisany, A. K. (1983) Teratogenicity in the rat of the venom from the scorpion Androctonus amoureuxi (Aud. &Sav.). Toxicon 21, 177-189. Osman, O. H., Ismail, M., El-Asmar, M. F. and Ibrahim, S. A. (1972) Effect on the rat uterus of the venom from the scc,rpion Leiurus quinquestriatus. Toxicon 10, 363-366. Silva, D. G. (1966) The ultrastructure of myometrium of the rat with special reference to the innervation. Anat. Rec. 158, 21-34. Stjernquist, M. and Owman, C. (1985) Cholinergic and adrenergic neural control of smooth muscle function in the non-pre[~ant rat uterine cervix. Acta Physiol. scand. 124, 429-436.