Toxlcon, 1975, Vol. 13, DP. 233-239 . Peraamon Press. Printed In Groat Hrltain.
PHARMACOLOGICAL STUDIES OF SCORPION (ANDROCTONUS AMOREUXI AUD. & SAV.) VENOM A. GHAZAL, M. ISMAIL, A. A. ABnI~RAi~IAx
Department of Pharmacology, Faculty of Pharmacy, University of Alexandria, Egypt
and M. F. EL-Asn~R
Department of Biochemistry, Faculty of Medicine, Ain-Shams University, Cairo, Egypt (Acceptedjorpu6lication 2 Jortuary 1975) A. GR"~"r, M, Iae~n., A. A. Asnst.-Rwmrax and M. F. Et-Asar~x. Pharmacological studies of scorpion (Androctorwa mnoreuxl Aud. do Sav.) venom. Toxlcort 13, ?53-259. 1g~S.--Venom from the scorpion Androctanus omoreuxi was fractionated into eight components by cellulose acetate electrophoresis, using barbitone buffer pH 8"6. OnIX one of the electrophoresic fractions was lethal in mice . The ln,o of the venom following i.m. mlecHon into mice was estimated to be 0" 88 f 0"06 mg per kg. The txrdiovascular effects of the venom appeared to be mediated through stimulation of both parts of the autonomic nervous system with predominance of pathetic stimulation, and release of tissue catecholamines. The venom markedly decreased tone of the spontaneously contracting rat uterus in all states of uterine function except is pregnancy whew it produced a stimulant effect. The uterine inhibitory effect of the venom is probably due to stimulation of the ß-adreaergicraoeptors as it was blocked by propranolol . The venom feat increased the amplitude and then blocked the twitches of the rat phrenic nerve--hemidiaphragm and caused contrachm and decrease of twitch height of the tlbialis anterior muscle preparation. Af the various fractions separated by cellulose acetate electrophoresis, only three fractions showed activity on the various preparations studied. INTRODUCTION
DasPTrB the widespread distribution of the scorpion Androctonus amorettxi (Aud. & Sav .)
in Egypt and neighbouring countries (BALOZBT, 1971), very few studies concerning its venom have been reported in the literature. This might be due in part to the greater interest shown, through the outstanding studies of Miranda and his group (M>RAxnA et al., 1964, 1966, 1970 ; RocHAT et al., 1970, 1972), in the venom from the closely related scorpion Androctonus australis . In view of the scanty information available concerning A. amoreuxi, it was of interest to investigate the pharmacological properties of its venom. MATF?RiAi s AND METHODS Venom was obtained from mature A. onwrruxl as described by Is~u, et al. (1973). Electrophoretic separation of the venom was performed in a horizontal Shandon tank using veronal buffer (005 ionic strength,pH 8"6) and cellulose acetato strips (Celagram, Shandon, London). Twenty-five ltl of dodo venom solution in distilled watea (20 mg per ml) were applied aloeg a 2"5 cm line marked on the paper. Elxtrophoresis was conducted at room temperature (22-25°C) for 140 min applying ZS V per cm. At the end of the run, the strips were dried in hot air and stained with Poncesu S (0"2 per cent in 3 per cent tricliloroacetic acid). For preparative electrophoresis 50 ltl samples of crude venom sohrtion were applied along a 6 cm line on cellulose acetate strips (78 x 150 mm). At the end of an electrophoretic run, a median longitudinal strip 1 cm wide was cut out and stained in order to locate the protein bands on the unstained sides. Protein beads were theme eluted from paper strips with 0"3 per cent NaCI solution . The concentration of the protein in the eluates was d ed apectro~hotometrically at 280 nm. The r.n of the crude venom was cTetert~med by the method of M>zl~rt and Tennait (1944) using 106 mice (20-30 g body weight). The toxicity of the protein fractions separated by electrophoresis of the crude 253 710YICON 1975 Yat. 13
254
A. GHAZAL, M. FSMAIL, A. A. ABDELrRAHMAN and M. F. EIrASMAR
venom, was detenninod in mice using 3 animals for each fraction. Generally 100 ug of protein were injected i.m . with the mice being observal for 24 hr and mortality, if any, recorded. Post mortem examinations were made. The isolated rabbit and guinea pig hearts (Langendorff preparation) were prepared as described by Is~,u, et al. (1973) . Blood pressure and respiration recordings were carried out mairily in cats, but also dogs, guinea pigs and rats were used. The methods were described previously (i3MAII. et al., 1973). The rat hindquarters reparation was performed as described by Buxx (1952) . The assessment of vascular permeabthty at e site of injection was carried out in rats by an adaptation of the method of Junex and WII.LOUGHHY (1962) . Estimation of dye leakage was made by the macroscopic appearance of the skin . The cat tibialis anterior muscle preparation (BxowN,1938) was prepared from cats of either sex (3-5 kg) anaesthetized with a mixture of chloralose (70 mg per kg) and pentobarbitone (5 mg per kg) injected intraperitoneally. The muscle was excited either once every 10 sec or once every sec by rectangular pulses (0~5 cosec duration) applied to the lateral popliteal nerve and using a stimulus strength of about 8 V. The venom was injected either intravenousl through a femoral vein or close-arterially into the anterior tibial artery. The isolated phrenic nerve-~emidiaphragm of tho rat (BtJi exuaa,1946) was mounted in Krebs-Henseleit solution (37°C). The muscle was excited either once every 10 sec or once every sec by rectangular pulses (200 psoc duration) applied to the phrenic nerve. The strength of the shocks was nearly twice that required to evoke a maximal twitch. The isolated spontaneously contracting rat uterus was removed and each horn was suspended separately in a 30 ml organ bath containing mammalian Ringer-Locke solution at 3T°C. Recordings wem made on a smoked drum with an isotonic lever with frontal writing point, adjusted to have a magnification of 4 and a constant load of 2 g. The isolated rat uterus prepared according to ns J.u .ox et al. (1975) and not showing any spontaneous contractions was also studied. The venom was also tested on the isolated rabbit's intestine and guinea pig ileum (MAONUS, 1904). RESULTS
Electrophoresis of the crude venom at pH 8"6 revealed 8 prominent bands which migrated towards the anode (Fig. 1). The l.nao of the crude venom injected intramuscularly into mice was 0"88 ~ 0"06 mg per kg. The venom caused paralysis at the site of injection extending to involve both hind limbs . The animals exhibited signs of irritation, biting the injected leg. Rapid respiration appeared followed by gasping and periodic breathing, tremors, incoordination of movements and convulsions. Death occurred within 10-20 min when using the venom in a dose of 2 mg per kg . Autopsy revealed no signs of bleeding. Injecting the various fractions separated by cellulose acetate electrophoresis into mice (100 Fig per mouse) showed that only fraction 4 was lethal. Fractions 2, 3, 5 and 8 caused paralysis at the site of injection followed by recovery within 1-8 hr. Fractions 6 and 7 as well as the band at the origin were without effect . Pharmacological investigations
The effects reported below are typical responses, each obtained in at least 6 similar experiments. The isolated heart
The typical effects of the venom on hearts obtained from 10 rabbits, 6 guinea pigs and 6 rats are shown in Fig. 2. They consisted of initial negative isotropic and chronotropic effects followed by a marked positive isotropic effect which lasted for more than 20 min. The heart rate, on the other hand, remained slower than before injection of the venom. These cardiac effects were produced by doses ranging between 250 and 500 Rg in case of rabbit's hearts and between 20 and 50 Kg in the case of rat and guinea pig hearts . Tachyphylaxis developed on repeating the injection of the venom. Treatment with nicotine or atropine blocked the initial cardiac depressant effect of the venom, while perfusion of the TOXICON 1975 Yob 13
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FIG. 2. EFFECT OF SCORPION VENOl1i ON THE LSOIATED RABBTr'S HEART .
~, Venom (500 ~~ ; ", acetylcholine (3 hg) ; O, atropine (300 ~ 3 min before injection of acetylcholine ; " , ~soprenaline (2 lIg) . At DS, the drum was stopped, and where the individual contractions are clearly seen, the drum sp~d was increased to 120 mm per min for S sec. Between (a) and b) propranolol was infused at a rate of 1 ug per min for 30 min.
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FIG. S. EFFECT OF SCORPION VENOM ON THE RAT PHRENIC NERVE-HEMII)IAPHRAGM PREPARATION.
Rat phrenic nerve-hemidiaphragm in Krebs-Henseleit stimulated at 0~1 Hz . ~, Venom (10 hg per ml); ~, tetanic stimulation of the phrenic nerve (SO Hz for 10 sec) . T'he downward deflections indicate contractions of the diaphragm. FIG. G. EFFECT OF SCORPION VENOM ON TFSB CAT TIHIALiS ANTkRiOR MUSCLE PREPARATION.
Female cats 3-3~5 kg under chloralose anaesthesia . Maximal twitches of the tibialis anterior muscle was elicited by stimulation of the nerve once every 10 sec in (a)and (c), and once everysec in (b). V, Venom ; F6, fraction S separated by cellulose acetate electrophoresis . The numbers indicate doses in micrograms injected close arterially at the solid circles . The documented deflections indicate contractions of the tibialis anterior muscle . TOX)CON 1975 Yol. 13
Pharniacological Studies of Scorpion (Androctonus anwreuxl Aud . & Sav .) Venom
233
heart with propranolol at a rate of 0~5-1~0 pg per min for 30 min before injection of the venom attenuated but incompletely blocked the positive inotropic effect of the venom. On hearts of reserpine-treated rabbits (5 animals), the venom caused bradycardia, and in most experiments this progressed to a temporary stoppage of the heart. The cardiac depressant effect of the venom was followed by a positive inotropic effect . Again, the depressant effect of the venom was blocked by treatment with nicotine or atropine while the stimulant action was attenuated but not blocked by treatment with propranolol. Testing the various fractions separated by cellulose acetate electrophoresis on the rabbit's heart (2501tg each), showed that fraction 4 had a cardiac stimulant effect, while the other fractions produced very slight depression. Bloodpressure and respiration recordings
These experiments were carried out on 15 cats, 2 dogs, 3 guinea pigs and 3 rats. In cats, the venom in a dose of 300 pg per kg produced a short lasting hypotension followed by a pronounced hypotensive response that lasted 10-15 min. Treatment with atropine (2 mg per kg) blocked the initial hypotensive effect of the venom and potentiated the subsequent hypertension. Repeating the injection of the venom showed evidence of tachyphylaxis. Carotid sinus and body denervation (4 cats) and/or bilateral vagotomy before or after carotid sinus and body denervation, markedly potentiated the hypotensive effect of the venom and prolonged its duration. The hypotensive effect was blocked by treatment with phenoxybenzamine or tolazoline (3 mg per kg). In two cats, pretreatment with phenoxybenzamine (3 mg per kg), 10 min before injecting the venom, blocked the hypotensive effect and clearly revealed the hypotensive action of the venom. The hypotensive effect was blocked by atropine treatment. In dogs, guinea pigs and rats, the venom produced effects similar to those produced in cats except that the hypotensive effect was not preceded by hypotension. The hypotensive effect was blocked by the a-sympatholytic drugs tolazoline or phenoxybenzamine . In the reserpine-treated cat (3 animals), where tyramine (5 mg per cat) was ineffective, the venom in a dose of 500 Rg per kg caused a hypotensive effect which was blocked by tolazoline. The injection of thevenom in cats resulted in a decreased rate ofrespiration accompanied by an increase in the depth of respiration. After repeated injections of the venom or injection of large doses (500-10001tg per kg), complete arrest of respiration occurred . In some cats periodic breathing was observed. Hindquarters perfusion and capillary permeability in rats A. amoreuxi venom in doses of 100-200 pg markedly reduced the perfusion rate in the
rat hindquarters preparation. The action started 1-2 min after injecting the venom, reached a peak (30-AS per cent reduction) within 5-10 min, and recovery was slow, taking 1-2 hr. The reduction in perfusion rate by the venom was blocked by injecting 1 mg phenoxybenzamine 10 min before injecting the venom. A. amoreuxi venom did not cause any significant increase in capillary permeability in the rat as shown by the degree of dye leakage into the skin 2 hr after injecting the venom. The isolated rat uterus
The action of the venom was studied on the following 4 physiological states : (1) natural oestrus (12 rats), (2) induced oestrus (9 rats) ; cestradiol dipropionate (2~5 mg per kg) was injected subcutaneously 25-45 hr before the experiment, (3) progestational phase (13 rats) ; T~OXICON 1971 Yot. 13
256
A. GHAZAL, M. ISMAIL, A. A. ABDEL-RAHMAN and M. F. EL-ASMAR
the rats were ovariectomized and treated with progesterone as described by TOZ~uLL (1965), and (4) pregnancy (14 rats) ; the uteri were taken on the 5th-10th day of pregnancy (early pregnancy) and on the 18th-21st day of pregnancy (late pregnancy) ; day one of pregnancy being determined by the appearance of vaginal plug. The crude venom in doses of 0~6-2~0 pg per ml markedly decreased the tone of the spontaneously contracting rat uterus in all states of uterine function with the exception of pregnancy, where it produced a stimulant effect . This uterine inhibitory effect was blocked by treatment with the ß-adrenergic blocking drug propranolol (0~5-1~0 ~g per ml) added 10 min before addition of the venom. In induced oestrus, following treatment with propranolol, the venom caused a stimulant action which was blocked by atropine (Fig. 3). This stimulant action of the venom was absent in uteri in natural oestrus and in the progestational phase (Fig. 4). The uterine stimulant effect of the venom in late pregnancy was blocked by atropine (1 Ftg per ml). Of the various fractions separated by cellulose acetate electrophoresis, only fractions 3 and 4 (2 pg per ml) showed an inhibitory effect . The effect, however, was much less than that produced by equal concentrations of the crude venom. A . amoreuxi venom in concentrations of 1-30 pg per ml did not cause any stimulation of the rat uterus in de Jalon solution ; however, the contractions produced by subsequent doses of serotonin and acetylcholine were reduced. Neuromuscular junctions On the rat phrenic nerve-hemidiaphragm preparation, the venom (10 pg per ml) produced a marked increase in the amplitude of twitches which reached a peak within 10-15 min. The increase in twitch height was followed by a gradual decrease of twitch activity, and almost complete block occurred within 20-60 min (Fig. ~. Tetanic stimulation of the phrenic nerve (50 Hz for 10 sec) during partial block produced by the venom showed that the tension of tetanus was well maintained . Testing the various fractions separated by cellulose acetate electrophoresis (10 ug per ml) showed that fractions 3 and 4 markedly increased the amplitude of twitches ; however, they produced no blocking effect . The other fractions were without any effect. In the cat tibialis anterior muscle preparation, the venom in intravenous doses of 100-1400 per kg caused a slight contracture and increase in the amplitude of twitches . When the Rg venom was injected close arterially in a dose of 50-100 pg per kg, it resulted in a marked contracture and decrease of muscle twitches which in most cases proceeded to complete blockade of twitch activity within 20-50 min (Fig. 6). When the muscle was stimulated at higher frequency (1 Hz), the contracture was more pronounced and the onset of contracture and block was more rapid and was produced by smaller doses of the venom (Fig. 6). Of the various fractions separated by cellulose acetate electrophoresis, only fraction 5 was active on the tibialis anterior muscle preparation and its effect was similar to that of the crude venom (Fig. 6). In both the phrenic nerve-hemidiaphragm preparation and the cat tibialis anterior muscle preparation, it was observed that following treatment with the venom, the relaxation after each contraction was much prolonged and in some cases required several seconds to be completed. The guinea pig ileuns and the rabbit intestine
In the guinea pig ileum, small doses of the venom (0~5-1 pg per ml) potentiated the
1'OXICON I97S Vol. I3
Pharmacological Studies of Scorpion (Mdroctonus mnoreuxt Aud. & Sav.) Venom
257
contractions produced by acetylcholine, while higher doses (more than 3 Rg per ml) produced a contraction which was blocked by nicotine (10 Kg per ml) or atropine (1 ~g per ml). In the rabbit intestine, the venom (1 ltg per ml) produced a powerful contraction which was attenuated by nicotine (10 pg per ml) and blocked by atropine (1 pg per ml). DISCUSSION
The cardiovascular effects of A . amoreuxi venom appear to be mediated through stimulation of both parts of the autonomic nervous system with predominance of sympathetic stimulation and release of tissue catecholamines ; effects which are similar to those of the venoms from the scorpions Leiurus quinquestriatus, Buthus minax and Pandinus excitialis (IsMnu, et al., 1972, 1973, 1974). However, some qualitative differences exist between the 4 venoms. Thus, the cardiac stimulant effect of A. amoreuxi venom was preceded by bradycardia, a property not possessed by the other 3 venoms . In this respect, the effect of A . amoreuxi venom resembles that from Tityus serrulatus on the guinea pig heart (CORRADO et al., 1968). In addition, a significant portion of the cardiac stimulant effect of A. amoreuxi venom appeared to be mediated through a direct mechanism as was evident from its resistance to blockade by propranolol. This property is possessed, though to a much lesser degree, by L. quinquestriatus and P. exitialis venoms (ISMAIL et al., 1972, 1974). The initial cardiac inhibitory action ofthe venom is probably mediated throughcholinergic stimulation, mainly ganglionic stimulation, as was evident from its blockade by nicotine. The cholinergic component of the venom was more clearly seen after depletion of the catecholamines stores by reserpine treatment. The effect of A. amoreuxi venom on arterial blood pressure is similar to that of other scorpion venoms (ISMAII, et a1.,1972, 1973, 1974). Scorpion venoms were shown to produce powerful contraction of the spontaneously contracting rat uterus partly by direct action and partly through the release of kinins, prostaglandins and/or slow reacting substances) (Os~tAx et al., 1972 ; Is~tnu. et al., 1974). A. amoreuxi venom, therefore, is peculiar in causing relaxation of the spontaneously contracting rat uterus. The effect appears to be mediated through stimulation of the ß-adrenergic receptors as was evident from its blockade by propranolol, a ß-adrenergic receptor blocking agent. It is known that the adrenergic receptors in the uterus are only of the ß-type (L$vx and Tozzi, 1963). A cholinergic component in the action of the venom (blocked by atropine) was seen in uteri obtained from pregnant rats, and following treatment with propranolol in uteri in induced oestrus. The cholinergic effect of the venom seems to be linked with the predominance of oestrogen action in the uterus . It is known that after oestrogen treatment and in late pregnancy the rat uterus is predominantly under the influence of oestrogens (MAA r .T. and MILLER, 1964; TOT'E~IILL, 1967; KNU~roN, 1968). Excitatory a-adrenergic receptors were shown to exist in these two physiological states alter blockade of the ß-adrenergic receptors with propranolol (TOTHILL, 1967; AEDEL-Azlz and HAgRY, 1972). However, this could not explain the stimulant action of the venom, as the venom caused stimulation of the late pregnancy uterus in the absence of propranolol. Similarly, in induced oestrus after treatment with propranolol, the stimulant action of the venom was shown at times where adrenaline failed to elicit any stimulation. Finally, the stimulant effect of the venom was not affected by phenoxybenzamine, an a-adrenergic receptor blocking agent, but was blocked with atropine . Serotonin which was reported to exist in many scorpion venoms (ADAbI and WE~ss, 1956, 1958) appears to be absent from A. amoreuxi venom as shown from the lack of effect on rat uterus in de Jalon solution . The inhibitory effect of the venom on the contractions ToxrcoN ~~rs vor. r3
238
A. GHAZAL, M. ISMAIL, A. A. ABDEL"RAHMAN and M. F. EIrASMAR
produced by subsequent addition of serotonin and acetylcholine might be due to its stimulating adrenergic receptors. The stimulant effect of the venom on the rabbit intestine and guinea pig ileum revealed the cholinergic component in its action, as evident from its blockade by nicotine and atropine. A . amoreuxi venom caused contracture and increase in the amplitude of twitches of the indirectly stimulated phrenic nerve-hemidiaphragm of the rat. The contracture was much more marked in the cat tibialis preparation but no increase in the twitch height was observed . In both preparations blockade of twitch activity eventually occurred. The effect on the hemidiaphragm of the rat is similar to that reported for L. quinquestriatus and B. nrinax venoms (ADAM and WEtss,1966 ; ISMAIL et al., 1973). It might be due to a slow depolarization by the venom of the excitable membrane of nerve and muscle, possibly due to an increase of sodium permeability and a delay in the inactivation of sodium permeability. The contraction produced in the cat tibialis anterior preparation is probably due to a direct action of the venom as it was present in denervated muscles and after neuromuscular blockade by Dtubocurarine (Ghazal, Ismail, Abdel-Rahmen and El-Asmar, unpublished observation) . Of the various fractions separated by cellulose acetate electrophoresis, fraction 4 was the only lethal fraction and in addition caused cardiac stimulation, uterine relaxation and an increase in the twitch height of the phrenic nerve hemidiaphragm of the rat. Our results are in agreement with that reported by ZLOTIüN et al. (1972) who performed starch gel zone electrophoresis on A. amoreuxi venom and demonstrated toxicity to mice in a cathodic fraction. Fraction 3 caused uterine relaxation and increased twitch height of the rat phrenic nerve-hemidiaphragm. Fraction 5 was only active on the cat tibialis anterior preparation. The other fractions were without effect on the different preparations studied. The difference in sensitivity of the rat phsenic nerve-hemidiaphragm and the cat tibialis anterior preparation towards fractions 3, 4 and 5 might reflect species variation. Acknowledgement-We are grateful to Mr. F. Wexi,FSS, of the British Museum (Natural History), London, England, for the identification of the scorpion Androctonus amoreuxi. REFERENCES AHn>urAztz, A. and BA%RY, N. (1972) The nature of the adrenoceptors in the post-pamrm rat uterus . Br.1. Pharmac. 4S, 304. Anew, K. R. and W~ C. (1936) 3-Hydroxytryptamine in swrpion venom. Nature, Lord 178, 421 . Anew, K. R . and Wars, C. (1938) The occurrence of 5-hydroxytryptamine in scorpion venom. J. exp. Biol. 35, 39 . Anew, K. R. and Wr:rss, C. (1966; published 1968) Some aspects of the pharmacology of the venoms of African scorpions. Meet s Inst . Butm#ma 33, 603. Ber.ozsr, L. (1971) Scorpionism in the Old World. In : Yeraomous Anfnwls mad tittir Yenoms, Vol. III, Venomous Invertebrates, p. 349, (BUCHERL, W. and Btrcra.sv, E., Eds.). New York : Academic Press. BROWN, G. L. (1938) Tho preparation of the tibialis anterior (cat) for close arterial injection. J. Phystol. Load. 92, 22P. Büt.HRnaa, E. (1946) Observations on the isolated phrenic nerve diaphragm preparation of the rat. Br. l. Pharnrac. Chemother. 1, 38 . BuRra, J. H. (1932) Biological Standardization. Oxford University Press. CoRReno, A. P., AN'PONIO, A. and Dnvrz, C. R. (1968) Brazilian scorpion venom (TFtyus serrulatus), an unusual sympathetic postganglionic stimulant . J. Pharmac. exp. 77rer. 164, 233. Isste>z., M., Ossux, O. H., IsRei~nrr, S. A. and ErrAsrreR, M. F. (1972) Cardiovascular and respiratory responses to the v~om from the scorpion Leittrrts grdrrquestriatus. E. .fir. med.1. 49, 273. I3MAIr., M ., Ossux, O. H. and Er,-Assure, M. F. (1973) Pharmacological studies of the venom from the scorpion Bathos minax (L. Koch). Toxicon 11, 13 . Issrea, M., Osrux, O. H., Guru,+, K. A. and KeRReR, M. A. (1974) Some pharmacological studies with scorpion (Pandinru excttialis) venom. Toxkon 12, 73. noxtcoN rgrs vor. rs
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259
ns J~r.ox, P. G., Bw BAYO, J. and ns J.+r.orv, M. G. (1945) Fartnacoterap . Actual. 3, 313. In : Pharmacological Experiments on Isolated Preparations, 1968, pp. 92-95, (Staff of Pharmacology Department, University of Edinburgh, Ed.) . Edinbwgh : Livingstone. Junwx, J. D. and Wn.r.oucr~, D. A. (1962) Quantitative method for the study of capillary permeability : extraction and determination oftrypan blue in tissues. J. Path. Bact. 83, 567. KNUrrox, A. (1968) The reactivity ofthe pregnant rat myometrium . J. Pharnr. Pharmac. 20, 569. Lsvst, H. and Tozzr, S. (1963) The adrenergrc receptive mechanism of the rat uterus. J. Pharrnac. exp. 77rer. 142, I78. M~ot~us, R (1904) PJlirgers Arch . ges Physiol. 103, 123. In : Pharmacological Experiments on Isolated Preparations, 1968, pp. 62-63, (Staff of Pharmacology Department, University of Edinbwgh, Ed.). Edinburgh: Livingstone . 1V1A4üi "1 i, J, M, and Mn.r.ete, M. D. (1464) Effects of metabolic inhibitors on the rat uterus and on its response to oxytocin . Am . J. Physiol. 206, 437. MII.t,~t, L. C. and T.~n~rrErr, M. L. (1944) Estimation of the ED6, and its error by means of logarithmicprobit graph paper. Proc . Soe. exp. Btol. Med. S7, 261 . Mnurro~, F., ROCHAT, H. and LrssrrzxY, S. (1964) Neurotoxins of two North African species of scorpions. II. Properties of neurotoxins (scorpamines) of Androctonus australis and Buthus occttanus. Toxicon 2, 113. Mntnrroe, F., ROCHAT, H., ROCI~IAT, C. and LrssrrzKV, S. (19C~ Molecular complexes in animal newotoxins . I. Scorpion venom newotoxins (Androctonus australis and Buthus occitanus) . Toxicon 4,123. Mnw~roe, F., Kurt:,r.~x, C., RocanT, H ., Rocx~r, C. and Lisstrzxv, S. (1970) Purification of animal neurotoxins . Isolation and characterization of eleven neurotoxins from the venoms of the scorpions Androctorws australis, Buthus oceitarws, and Leiurus quinquestriatus. Eur. J. Biochem. 16, 514. Osas~,rv, O. H., Law n., M., ErrAs~rt, M. F. and IHR~r, S. A. (1972) Effect on the rat uterus of the venom from the scorpion Lelurus quinquestriatus . Toxicon 10, 363. Roca+r, H ., Rocxnr, C., Mnterme, F., LtssrrzaY, S. and Enrux, P. (1970) The amino acid sequence of Neurotoxin I of Androctomrs australis Hector. Eör. J. Biochem. 17, 262. ROCHAT, H., ROCHAT, C., SAMPIERI, F. and Mnuxnx, F. (1972) The amino acid sequence of Neurotoxin II of Androctonus australLs Hector . Eur. J. Biochem. 28, 381 . Tozmz.L, A. (1965) The effect of four monamine oxidase inhibitors on the rat uterus . Br . J. Pharmac. Chemother. 25, 217 . TOT~a., A. (1967) Investigation of adrenaline reversal in the rat uterus by the induction of resistance to isoprenaline. Br. J. Pharmac. 29, 291. Zro~xar, E., M>R.~riDw, F. and LLYSr1ZKY, S. (1972) Proteins in scorpion venoms toxic to mammals and insects. Toxicon 10, 207.
zoxtcowigrs v~t. is
PHARMACOLOGICAL STUDIES OF SCORPION (ANDROCTONUS AMOREUXI AUD. & SAV.) VENOM A. GHAZAL, M. ISMAIL, A. A. ABnI~RAi~IAx
Department of Pharmacology, Faculty of Pharmacy, University of Alexandria, Egypt
and M. F. EL-Asn~R
Department of Biochemistry, Faculty of Medicine, Ain-Shams University, Cairo, Egypt (Acceptedjorpu6lication 2 Jortuary 1975) A. GR"~"r, M, Iae~n., A. A. Asnst.-Rwmrax and M. F. Et-Asar~x. Pharmacological studies of scorpion (Androctorwa mnoreuxl Aud. do Sav.) venom. Toxlcort 13, ?53-259. 1g~S.--Venom from the scorpion Androctanus omoreuxi was fractionated into eight components by cellulose acetate electrophoresis, using barbitone buffer pH 8"6. OnIX one of the electrophoresic fractions was lethal in mice . The ln,o of the venom following i.m. mlecHon into mice was estimated to be 0" 88 f 0"06 mg per kg. The txrdiovascular effects of the venom appeared to be mediated through stimulation of both parts of the autonomic nervous system with predominance of pathetic stimulation, and release of tissue catecholamines. The venom markedly decreased tone of the spontaneously contracting rat uterus in all states of uterine function except is pregnancy whew it produced a stimulant effect. The uterine inhibitory effect of the venom is probably due to stimulation of the ß-adreaergicraoeptors as it was blocked by propranolol . The venom feat increased the amplitude and then blocked the twitches of the rat phrenic nerve--hemidiaphragm and caused contrachm and decrease of twitch height of the tlbialis anterior muscle preparation. Af the various fractions separated by cellulose acetate electrophoresis, only three fractions showed activity on the various preparations studied. INTRODUCTION
DasPTrB the widespread distribution of the scorpion Androctonus amorettxi (Aud. & Sav .)
in Egypt and neighbouring countries (BALOZBT, 1971), very few studies concerning its venom have been reported in the literature. This might be due in part to the greater interest shown, through the outstanding studies of Miranda and his group (M>RAxnA et al., 1964, 1966, 1970 ; RocHAT et al., 1970, 1972), in the venom from the closely related scorpion Androctonus australis . In view of the scanty information available concerning A. amoreuxi, it was of interest to investigate the pharmacological properties of its venom. MATF?RiAi s AND METHODS Venom was obtained from mature A. onwrruxl as described by Is~u, et al. (1973). Electrophoretic separation of the venom was performed in a horizontal Shandon tank using veronal buffer (005 ionic strength,pH 8"6) and cellulose acetato strips (Celagram, Shandon, London). Twenty-five ltl of dodo venom solution in distilled watea (20 mg per ml) were applied aloeg a 2"5 cm line marked on the paper. Elxtrophoresis was conducted at room temperature (22-25°C) for 140 min applying ZS V per cm. At the end of the run, the strips were dried in hot air and stained with Poncesu S (0"2 per cent in 3 per cent tricliloroacetic acid). For preparative electrophoresis 50 ltl samples of crude venom sohrtion were applied along a 6 cm line on cellulose acetate strips (78 x 150 mm). At the end of an electrophoretic run, a median longitudinal strip 1 cm wide was cut out and stained in order to locate the protein bands on the unstained sides. Protein beads were theme eluted from paper strips with 0"3 per cent NaCI solution . The concentration of the protein in the eluates was d ed apectro~hotometrically at 280 nm. The r.n of the crude venom was cTetert~med by the method of M>zl~rt and Tennait (1944) using 106 mice (20-30 g body weight). The toxicity of the protein fractions separated by electrophoresis of the crude 253 710YICON 1975 Yat. 13
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A. GHAZAL, M. FSMAIL, A. A. ABDELrRAHMAN and M. F. EIrASMAR
venom, was detenninod in mice using 3 animals for each fraction. Generally 100 ug of protein were injected i.m . with the mice being observal for 24 hr and mortality, if any, recorded. Post mortem examinations were made. The isolated rabbit and guinea pig hearts (Langendorff preparation) were prepared as described by Is~,u, et al. (1973) . Blood pressure and respiration recordings were carried out mairily in cats, but also dogs, guinea pigs and rats were used. The methods were described previously (i3MAII. et al., 1973). The rat hindquarters reparation was performed as described by Buxx (1952) . The assessment of vascular permeabthty at e site of injection was carried out in rats by an adaptation of the method of Junex and WII.LOUGHHY (1962) . Estimation of dye leakage was made by the macroscopic appearance of the skin . The cat tibialis anterior muscle preparation (BxowN,1938) was prepared from cats of either sex (3-5 kg) anaesthetized with a mixture of chloralose (70 mg per kg) and pentobarbitone (5 mg per kg) injected intraperitoneally. The muscle was excited either once every 10 sec or once every sec by rectangular pulses (0~5 cosec duration) applied to the lateral popliteal nerve and using a stimulus strength of about 8 V. The venom was injected either intravenousl through a femoral vein or close-arterially into the anterior tibial artery. The isolated phrenic nerve-~emidiaphragm of tho rat (BtJi exuaa,1946) was mounted in Krebs-Henseleit solution (37°C). The muscle was excited either once every 10 sec or once every sec by rectangular pulses (200 psoc duration) applied to the phrenic nerve. The strength of the shocks was nearly twice that required to evoke a maximal twitch. The isolated spontaneously contracting rat uterus was removed and each horn was suspended separately in a 30 ml organ bath containing mammalian Ringer-Locke solution at 3T°C. Recordings wem made on a smoked drum with an isotonic lever with frontal writing point, adjusted to have a magnification of 4 and a constant load of 2 g. The isolated rat uterus prepared according to ns J.u .ox et al. (1975) and not showing any spontaneous contractions was also studied. The venom was also tested on the isolated rabbit's intestine and guinea pig ileum (MAONUS, 1904). RESULTS
Electrophoresis of the crude venom at pH 8"6 revealed 8 prominent bands which migrated towards the anode (Fig. 1). The l.nao of the crude venom injected intramuscularly into mice was 0"88 ~ 0"06 mg per kg. The venom caused paralysis at the site of injection extending to involve both hind limbs . The animals exhibited signs of irritation, biting the injected leg. Rapid respiration appeared followed by gasping and periodic breathing, tremors, incoordination of movements and convulsions. Death occurred within 10-20 min when using the venom in a dose of 2 mg per kg . Autopsy revealed no signs of bleeding. Injecting the various fractions separated by cellulose acetate electrophoresis into mice (100 Fig per mouse) showed that only fraction 4 was lethal. Fractions 2, 3, 5 and 8 caused paralysis at the site of injection followed by recovery within 1-8 hr. Fractions 6 and 7 as well as the band at the origin were without effect . Pharmacological investigations
The effects reported below are typical responses, each obtained in at least 6 similar experiments. The isolated heart
The typical effects of the venom on hearts obtained from 10 rabbits, 6 guinea pigs and 6 rats are shown in Fig. 2. They consisted of initial negative isotropic and chronotropic effects followed by a marked positive isotropic effect which lasted for more than 20 min. The heart rate, on the other hand, remained slower than before injection of the venom. These cardiac effects were produced by doses ranging between 250 and 500 Rg in case of rabbit's hearts and between 20 and 50 Kg in the case of rat and guinea pig hearts . Tachyphylaxis developed on repeating the injection of the venom. Treatment with nicotine or atropine blocked the initial cardiac depressant effect of the venom, while perfusion of the TOXICON 1975 Yob 13
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FIG. 2. EFFECT OF SCORPION VENOl1i ON THE LSOIATED RABBTr'S HEART .
~, Venom (500 ~~ ; ", acetylcholine (3 hg) ; O, atropine (300 ~ 3 min before injection of acetylcholine ; " , ~soprenaline (2 lIg) . At DS, the drum was stopped, and where the individual contractions are clearly seen, the drum sp~d was increased to 120 mm per min for S sec. Between (a) and b) propranolol was infused at a rate of 1 ug per min for 30 min.
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FIG. S. EFFECT OF SCORPION VENOM ON THE RAT PHRENIC NERVE-HEMII)IAPHRAGM PREPARATION.
Rat phrenic nerve-hemidiaphragm in Krebs-Henseleit stimulated at 0~1 Hz . ~, Venom (10 hg per ml); ~, tetanic stimulation of the phrenic nerve (SO Hz for 10 sec) . T'he downward deflections indicate contractions of the diaphragm. FIG. G. EFFECT OF SCORPION VENOM ON TFSB CAT TIHIALiS ANTkRiOR MUSCLE PREPARATION.
Female cats 3-3~5 kg under chloralose anaesthesia . Maximal twitches of the tibialis anterior muscle was elicited by stimulation of the nerve once every 10 sec in (a)and (c), and once everysec in (b). V, Venom ; F6, fraction S separated by cellulose acetate electrophoresis . The numbers indicate doses in micrograms injected close arterially at the solid circles . The documented deflections indicate contractions of the tibialis anterior muscle . TOX)CON 1975 Yol. 13
Pharniacological Studies of Scorpion (Androctonus anwreuxl Aud . & Sav .) Venom
233
heart with propranolol at a rate of 0~5-1~0 pg per min for 30 min before injection of the venom attenuated but incompletely blocked the positive inotropic effect of the venom. On hearts of reserpine-treated rabbits (5 animals), the venom caused bradycardia, and in most experiments this progressed to a temporary stoppage of the heart. The cardiac depressant effect of the venom was followed by a positive inotropic effect . Again, the depressant effect of the venom was blocked by treatment with nicotine or atropine while the stimulant action was attenuated but not blocked by treatment with propranolol. Testing the various fractions separated by cellulose acetate electrophoresis on the rabbit's heart (2501tg each), showed that fraction 4 had a cardiac stimulant effect, while the other fractions produced very slight depression. Bloodpressure and respiration recordings
These experiments were carried out on 15 cats, 2 dogs, 3 guinea pigs and 3 rats. In cats, the venom in a dose of 300 pg per kg produced a short lasting hypotension followed by a pronounced hypotensive response that lasted 10-15 min. Treatment with atropine (2 mg per kg) blocked the initial hypotensive effect of the venom and potentiated the subsequent hypertension. Repeating the injection of the venom showed evidence of tachyphylaxis. Carotid sinus and body denervation (4 cats) and/or bilateral vagotomy before or after carotid sinus and body denervation, markedly potentiated the hypotensive effect of the venom and prolonged its duration. The hypotensive effect was blocked by treatment with phenoxybenzamine or tolazoline (3 mg per kg). In two cats, pretreatment with phenoxybenzamine (3 mg per kg), 10 min before injecting the venom, blocked the hypotensive effect and clearly revealed the hypotensive action of the venom. The hypotensive effect was blocked by atropine treatment. In dogs, guinea pigs and rats, the venom produced effects similar to those produced in cats except that the hypotensive effect was not preceded by hypotension. The hypotensive effect was blocked by the a-sympatholytic drugs tolazoline or phenoxybenzamine . In the reserpine-treated cat (3 animals), where tyramine (5 mg per cat) was ineffective, the venom in a dose of 500 Rg per kg caused a hypotensive effect which was blocked by tolazoline. The injection of thevenom in cats resulted in a decreased rate ofrespiration accompanied by an increase in the depth of respiration. After repeated injections of the venom or injection of large doses (500-10001tg per kg), complete arrest of respiration occurred . In some cats periodic breathing was observed. Hindquarters perfusion and capillary permeability in rats A. amoreuxi venom in doses of 100-200 pg markedly reduced the perfusion rate in the
rat hindquarters preparation. The action started 1-2 min after injecting the venom, reached a peak (30-AS per cent reduction) within 5-10 min, and recovery was slow, taking 1-2 hr. The reduction in perfusion rate by the venom was blocked by injecting 1 mg phenoxybenzamine 10 min before injecting the venom. A. amoreuxi venom did not cause any significant increase in capillary permeability in the rat as shown by the degree of dye leakage into the skin 2 hr after injecting the venom. The isolated rat uterus
The action of the venom was studied on the following 4 physiological states : (1) natural oestrus (12 rats), (2) induced oestrus (9 rats) ; cestradiol dipropionate (2~5 mg per kg) was injected subcutaneously 25-45 hr before the experiment, (3) progestational phase (13 rats) ; T~OXICON 1971 Yot. 13
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A. GHAZAL, M. ISMAIL, A. A. ABDEL-RAHMAN and M. F. EL-ASMAR
the rats were ovariectomized and treated with progesterone as described by TOZ~uLL (1965), and (4) pregnancy (14 rats) ; the uteri were taken on the 5th-10th day of pregnancy (early pregnancy) and on the 18th-21st day of pregnancy (late pregnancy) ; day one of pregnancy being determined by the appearance of vaginal plug. The crude venom in doses of 0~6-2~0 pg per ml markedly decreased the tone of the spontaneously contracting rat uterus in all states of uterine function with the exception of pregnancy, where it produced a stimulant effect . This uterine inhibitory effect was blocked by treatment with the ß-adrenergic blocking drug propranolol (0~5-1~0 ~g per ml) added 10 min before addition of the venom. In induced oestrus, following treatment with propranolol, the venom caused a stimulant action which was blocked by atropine (Fig. 3). This stimulant action of the venom was absent in uteri in natural oestrus and in the progestational phase (Fig. 4). The uterine stimulant effect of the venom in late pregnancy was blocked by atropine (1 Ftg per ml). Of the various fractions separated by cellulose acetate electrophoresis, only fractions 3 and 4 (2 pg per ml) showed an inhibitory effect . The effect, however, was much less than that produced by equal concentrations of the crude venom. A . amoreuxi venom in concentrations of 1-30 pg per ml did not cause any stimulation of the rat uterus in de Jalon solution ; however, the contractions produced by subsequent doses of serotonin and acetylcholine were reduced. Neuromuscular junctions On the rat phrenic nerve-hemidiaphragm preparation, the venom (10 pg per ml) produced a marked increase in the amplitude of twitches which reached a peak within 10-15 min. The increase in twitch height was followed by a gradual decrease of twitch activity, and almost complete block occurred within 20-60 min (Fig. ~. Tetanic stimulation of the phrenic nerve (50 Hz for 10 sec) during partial block produced by the venom showed that the tension of tetanus was well maintained . Testing the various fractions separated by cellulose acetate electrophoresis (10 ug per ml) showed that fractions 3 and 4 markedly increased the amplitude of twitches ; however, they produced no blocking effect . The other fractions were without any effect. In the cat tibialis anterior muscle preparation, the venom in intravenous doses of 100-1400 per kg caused a slight contracture and increase in the amplitude of twitches . When the Rg venom was injected close arterially in a dose of 50-100 pg per kg, it resulted in a marked contracture and decrease of muscle twitches which in most cases proceeded to complete blockade of twitch activity within 20-50 min (Fig. 6). When the muscle was stimulated at higher frequency (1 Hz), the contracture was more pronounced and the onset of contracture and block was more rapid and was produced by smaller doses of the venom (Fig. 6). Of the various fractions separated by cellulose acetate electrophoresis, only fraction 5 was active on the tibialis anterior muscle preparation and its effect was similar to that of the crude venom (Fig. 6). In both the phrenic nerve-hemidiaphragm preparation and the cat tibialis anterior muscle preparation, it was observed that following treatment with the venom, the relaxation after each contraction was much prolonged and in some cases required several seconds to be completed. The guinea pig ileuns and the rabbit intestine
In the guinea pig ileum, small doses of the venom (0~5-1 pg per ml) potentiated the
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257
contractions produced by acetylcholine, while higher doses (more than 3 Rg per ml) produced a contraction which was blocked by nicotine (10 Kg per ml) or atropine (1 ~g per ml). In the rabbit intestine, the venom (1 ltg per ml) produced a powerful contraction which was attenuated by nicotine (10 pg per ml) and blocked by atropine (1 pg per ml). DISCUSSION
The cardiovascular effects of A . amoreuxi venom appear to be mediated through stimulation of both parts of the autonomic nervous system with predominance of sympathetic stimulation and release of tissue catecholamines ; effects which are similar to those of the venoms from the scorpions Leiurus quinquestriatus, Buthus minax and Pandinus excitialis (IsMnu, et al., 1972, 1973, 1974). However, some qualitative differences exist between the 4 venoms. Thus, the cardiac stimulant effect of A. amoreuxi venom was preceded by bradycardia, a property not possessed by the other 3 venoms . In this respect, the effect of A . amoreuxi venom resembles that from Tityus serrulatus on the guinea pig heart (CORRADO et al., 1968). In addition, a significant portion of the cardiac stimulant effect of A. amoreuxi venom appeared to be mediated through a direct mechanism as was evident from its resistance to blockade by propranolol. This property is possessed, though to a much lesser degree, by L. quinquestriatus and P. exitialis venoms (ISMAIL et al., 1972, 1974). The initial cardiac inhibitory action ofthe venom is probably mediated throughcholinergic stimulation, mainly ganglionic stimulation, as was evident from its blockade by nicotine. The cholinergic component of the venom was more clearly seen after depletion of the catecholamines stores by reserpine treatment. The effect of A. amoreuxi venom on arterial blood pressure is similar to that of other scorpion venoms (ISMAII, et a1.,1972, 1973, 1974). Scorpion venoms were shown to produce powerful contraction of the spontaneously contracting rat uterus partly by direct action and partly through the release of kinins, prostaglandins and/or slow reacting substances) (Os~tAx et al., 1972 ; Is~tnu. et al., 1974). A. amoreuxi venom, therefore, is peculiar in causing relaxation of the spontaneously contracting rat uterus. The effect appears to be mediated through stimulation of the ß-adrenergic receptors as was evident from its blockade by propranolol, a ß-adrenergic receptor blocking agent. It is known that the adrenergic receptors in the uterus are only of the ß-type (L$vx and Tozzi, 1963). A cholinergic component in the action of the venom (blocked by atropine) was seen in uteri obtained from pregnant rats, and following treatment with propranolol in uteri in induced oestrus. The cholinergic effect of the venom seems to be linked with the predominance of oestrogen action in the uterus . It is known that after oestrogen treatment and in late pregnancy the rat uterus is predominantly under the influence of oestrogens (MAA r .T. and MILLER, 1964; TOT'E~IILL, 1967; KNU~roN, 1968). Excitatory a-adrenergic receptors were shown to exist in these two physiological states alter blockade of the ß-adrenergic receptors with propranolol (TOTHILL, 1967; AEDEL-Azlz and HAgRY, 1972). However, this could not explain the stimulant action of the venom, as the venom caused stimulation of the late pregnancy uterus in the absence of propranolol. Similarly, in induced oestrus after treatment with propranolol, the stimulant action of the venom was shown at times where adrenaline failed to elicit any stimulation. Finally, the stimulant effect of the venom was not affected by phenoxybenzamine, an a-adrenergic receptor blocking agent, but was blocked with atropine . Serotonin which was reported to exist in many scorpion venoms (ADAbI and WE~ss, 1956, 1958) appears to be absent from A. amoreuxi venom as shown from the lack of effect on rat uterus in de Jalon solution . The inhibitory effect of the venom on the contractions ToxrcoN ~~rs vor. r3
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A. GHAZAL, M. ISMAIL, A. A. ABDEL"RAHMAN and M. F. EIrASMAR
produced by subsequent addition of serotonin and acetylcholine might be due to its stimulating adrenergic receptors. The stimulant effect of the venom on the rabbit intestine and guinea pig ileum revealed the cholinergic component in its action, as evident from its blockade by nicotine and atropine. A . amoreuxi venom caused contracture and increase in the amplitude of twitches of the indirectly stimulated phrenic nerve-hemidiaphragm of the rat. The contracture was much more marked in the cat tibialis preparation but no increase in the twitch height was observed . In both preparations blockade of twitch activity eventually occurred. The effect on the hemidiaphragm of the rat is similar to that reported for L. quinquestriatus and B. nrinax venoms (ADAM and WEtss,1966 ; ISMAIL et al., 1973). It might be due to a slow depolarization by the venom of the excitable membrane of nerve and muscle, possibly due to an increase of sodium permeability and a delay in the inactivation of sodium permeability. The contraction produced in the cat tibialis anterior preparation is probably due to a direct action of the venom as it was present in denervated muscles and after neuromuscular blockade by Dtubocurarine (Ghazal, Ismail, Abdel-Rahmen and El-Asmar, unpublished observation) . Of the various fractions separated by cellulose acetate electrophoresis, fraction 4 was the only lethal fraction and in addition caused cardiac stimulation, uterine relaxation and an increase in the twitch height of the phrenic nerve hemidiaphragm of the rat. Our results are in agreement with that reported by ZLOTIüN et al. (1972) who performed starch gel zone electrophoresis on A. amoreuxi venom and demonstrated toxicity to mice in a cathodic fraction. Fraction 3 caused uterine relaxation and increased twitch height of the rat phrenic nerve-hemidiaphragm. Fraction 5 was only active on the cat tibialis anterior preparation. The other fractions were without effect on the different preparations studied. The difference in sensitivity of the rat phsenic nerve-hemidiaphragm and the cat tibialis anterior preparation towards fractions 3, 4 and 5 might reflect species variation. Acknowledgement-We are grateful to Mr. F. Wexi,FSS, of the British Museum (Natural History), London, England, for the identification of the scorpion Androctonus amoreuxi. REFERENCES AHn>urAztz, A. and BA%RY, N. (1972) The nature of the adrenoceptors in the post-pamrm rat uterus . Br.1. Pharmac. 4S, 304. Anew, K. R. and W~ C. (1936) 3-Hydroxytryptamine in swrpion venom. Nature, Lord 178, 421 . Anew, K. R . and Wars, C. (1938) The occurrence of 5-hydroxytryptamine in scorpion venom. J. exp. Biol. 35, 39 . Anew, K. R. and Wr:rss, C. (1966; published 1968) Some aspects of the pharmacology of the venoms of African scorpions. Meet s Inst . Butm#ma 33, 603. Ber.ozsr, L. (1971) Scorpionism in the Old World. In : Yeraomous Anfnwls mad tittir Yenoms, Vol. III, Venomous Invertebrates, p. 349, (BUCHERL, W. and Btrcra.sv, E., Eds.). New York : Academic Press. BROWN, G. L. (1938) Tho preparation of the tibialis anterior (cat) for close arterial injection. J. Phystol. Load. 92, 22P. Büt.HRnaa, E. (1946) Observations on the isolated phrenic nerve diaphragm preparation of the rat. Br. l. Pharnrac. Chemother. 1, 38 . BuRra, J. H. (1932) Biological Standardization. Oxford University Press. CoRReno, A. P., AN'PONIO, A. and Dnvrz, C. R. (1968) Brazilian scorpion venom (TFtyus serrulatus), an unusual sympathetic postganglionic stimulant . J. Pharmac. exp. 77rer. 164, 233. Isste>z., M., Ossux, O. H., IsRei~nrr, S. A. and ErrAsrreR, M. F. (1972) Cardiovascular and respiratory responses to the v~om from the scorpion Leittrrts grdrrquestriatus. E. .fir. med.1. 49, 273. I3MAIr., M ., Ossux, O. H. and Er,-Assure, M. F. (1973) Pharmacological studies of the venom from the scorpion Bathos minax (L. Koch). Toxicon 11, 13 . Issrea, M., Osrux, O. H., Guru,+, K. A. and KeRReR, M. A. (1974) Some pharmacological studies with scorpion (Pandinru excttialis) venom. Toxkon 12, 73. noxtcoN rgrs vor. rs
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ns J~r.ox, P. G., Bw BAYO, J. and ns J.+r.orv, M. G. (1945) Fartnacoterap . Actual. 3, 313. In : Pharmacological Experiments on Isolated Preparations, 1968, pp. 92-95, (Staff of Pharmacology Department, University of Edinburgh, Ed.) . Edinbwgh : Livingstone. Junwx, J. D. and Wn.r.oucr~, D. A. (1962) Quantitative method for the study of capillary permeability : extraction and determination oftrypan blue in tissues. J. Path. Bact. 83, 567. KNUrrox, A. (1968) The reactivity ofthe pregnant rat myometrium . J. Pharnr. Pharmac. 20, 569. Lsvst, H. and Tozzr, S. (1963) The adrenergrc receptive mechanism of the rat uterus. J. Pharrnac. exp. 77rer. 142, I78. M~ot~us, R (1904) PJlirgers Arch . ges Physiol. 103, 123. In : Pharmacological Experiments on Isolated Preparations, 1968, pp. 62-63, (Staff of Pharmacology Department, University of Edinbwgh, Ed.). Edinburgh: Livingstone . 1V1A4üi "1 i, J, M, and Mn.r.ete, M. D. (1464) Effects of metabolic inhibitors on the rat uterus and on its response to oxytocin . Am . J. Physiol. 206, 437. MII.t,~t, L. C. and T.~n~rrErr, M. L. (1944) Estimation of the ED6, and its error by means of logarithmicprobit graph paper. Proc . Soe. exp. Btol. Med. S7, 261 . Mnurro~, F., ROCHAT, H. and LrssrrzxY, S. (1964) Neurotoxins of two North African species of scorpions. II. Properties of neurotoxins (scorpamines) of Androctonus australis and Buthus occttanus. Toxicon 2, 113. Mntnrroe, F., ROCHAT, H., ROCI~IAT, C. and LrssrrzKV, S. (19C~ Molecular complexes in animal newotoxins . I. Scorpion venom newotoxins (Androctonus australis and Buthus occitanus) . Toxicon 4,123. Mnw~roe, F., Kurt:,r.~x, C., RocanT, H ., Rocx~r, C. and Lisstrzxv, S. (1970) Purification of animal neurotoxins . Isolation and characterization of eleven neurotoxins from the venoms of the scorpions Androctorws australis, Buthus oceitarws, and Leiurus quinquestriatus. Eur. J. Biochem. 16, 514. Osas~,rv, O. H., Law n., M., ErrAs~rt, M. F. and IHR~r, S. A. (1972) Effect on the rat uterus of the venom from the scorpion Lelurus quinquestriatus . Toxicon 10, 363. Roca+r, H ., Rocxnr, C., Mnterme, F., LtssrrzaY, S. and Enrux, P. (1970) The amino acid sequence of Neurotoxin I of Androctomrs australis Hector. Eör. J. Biochem. 17, 262. ROCHAT, H., ROCHAT, C., SAMPIERI, F. and Mnuxnx, F. (1972) The amino acid sequence of Neurotoxin II of Androctonus australLs Hector . Eur. J. Biochem. 28, 381 . Tozmz.L, A. (1965) The effect of four monamine oxidase inhibitors on the rat uterus . Br . J. Pharmac. Chemother. 25, 217 . TOT~a., A. (1967) Investigation of adrenaline reversal in the rat uterus by the induction of resistance to isoprenaline. Br. J. Pharmac. 29, 291. Zro~xar, E., M>R.~riDw, F. and LLYSr1ZKY, S. (1972) Proteins in scorpion venoms toxic to mammals and insects. Toxicon 10, 207.
zoxtcowigrs v~t. is
