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The action of toxins derived from scorpion venom on the ileal smooth muscle preparation
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THE ACTION OF TOXINS DERIVED FROM SCORPION VENOM ON THE ILEAL SMOOTH MUSCLE PREPARATION* MEC,vua T1NTPU1,vEx,t Tax Z>~uct~ and F.~.uHV ZtoTglrt Department of Entomology and Venomous Anfmals, The Hebrew University of Jerusalem, Jerusalem, Israel (Accepted for prrbltrntlon
9
March
197
M. Tnvlruc vBa, T . Z8RAC8IA and B. Zwr~r. The action of toxins derived from scorpion venom on the deal smooth muscle preparation. Toxicon 14, 371-377,1976.-The'inse~t','cxuatacean' and `mammal' I end II toxins previously purified from tho venom of the scorpion Androcttmrct arratm/Lv Hector according to their fiy larvae paralysis, isopoda paralysis and mice lethality, r+espectlvely, were applied on the guinea pig ilaim smooth muscle preparation and it has bees found that : (1) tho 'crustacean' toxin induced a sustained-prolonged contracdon is contrast to the rhythmic spasmodic deal behaviour caused by the crude venom and the `mammal' toxins. (2) The excitatory effect of these substances wan due to a postganglionic ptraynaptic stimulation rosultin8 in the release of a cholinergic transmitter. (3) Prolonged application ofthe 'mammal' as well as `crustacean' toxins, caused e depression ofpostsynaptic reactivity as observed in a reduced deal response to several agonists. (4) The 'insect' toxin, wan inactive in the t7eum smooth musde preparation. INTRODUCTION Wirx T~ aid of simple bioassays, based on lethality or paralysis of test animals, it has been shown that the activity of scorpion venom on arthropods is due to specific factors (Ztarx~ et al., 1971, 1972a, b), that differ from the well-known toxins active on mammals (MIRANDA et al., 1964, 1970; ROCx~.T et al., 1967, 1970) . Furthermore, the paralysis and death of isopods (Crustacea) by A. australis venom is due to a protein factor different from the one causing paralysis and death of insects (ZLOTSIN et al., 1972b, 1975) . The small dosage, speed of paralysis and the symptomatology indicate that these effects are probably due to a neurotoxic action of these specific factors. These materials have also been tested in a more specific fashion : The `insect' toxin (ZLO~rxua et al., 1971) blocked synaptic transmission in the sixth abdominal ganglion of the central nervous system of Periplaneta amerlcana (n'Arns.to et al., 1972). The most potent `mammal' toxin (Tx In isolated from the venom of the same scorpion (MIRANDA et al., 1970) was inactive. Likewise, the `crustacean' toxin fraction induced an excitatory blockade of the crayfish stretch receptor (PANSA et al., 1973) while Tx II and the `.insect' toxin were ineffective. Thus, it has been assumed that the specificity of the above toxins is based on their specific affinity for neural systems of the relevant groups of organisms . It seemed pertinent to perform an examination of the arthropod toxins by applying them to a vertebrate system. The smooth muscle preparation of the guinea pig ileum has 'Supported by orant Nb 730, from the U.S.-Israel Binational Scieaoe Foundation. tPtesent address : Queens University, Dept. Biology, Kingston, Canada K7L 3N6. 371 710XICON 1976 Yol. I ~
372
MELVII~i TiNTPULVER, TAMAR ZERACHIA aad Ei .iAHÜ ZhOTKiN
been used to test the action of scorpion venoms and toxins (PATr~sox,1962 ; FRaut>ß-NL~tA and Du~iz, 1970 ; CuxtiA MEio et al., 1973), thus supplying a basis for comparison with our present studies. MA~~ Q AND METHODS Crude venom from Androctonur xuatralla Hector was electrically milked from the ecorpiona and subsequenUy lyophilized. The 'mammal' toxins I and II, `insect toxin' (iT1, and 'crustacean' toxin (CT) were purißed from the same venom (M~~~ et x1 .,1970 ; Ztar~ et x1.,1971,1976) . The following drugs wore used : acetyk:holine chloride, atropine sulphate, physostigmine (eserine) sulphate, diplrenhydtamino hydrochloride (Benadryl), hexamethonium bromide, S-hydroxy-tryptamiae creatinine sulphate (saotonin), nicotine sulphate and histamine dihydrochloride, all obtained from Sigma Co . ; morphine sulphate (Merck Co.) ; methyaergide maleate (Deaeril) (Saadoz Co.) ; tetrodotoxin citrate (Sanlgo--C~lbiocheai). All chemicals wero of analytical grade. The appropriate Tyrode solution was composed of (g per 1) : NaC18~0; KC10~2 ; MgC12 0-0S ; CaClz 0"2; NaH2P04 005; NaHC031-0 and glucose 1-0 (Howeux et x1.,1972). Starved male guinea Pigs were killed by a blow an the head. The ileum was removed, washed and segmenta of 2-3 cm were sectioned and susp~ded in a 15 ml organ bath containing seratod Tyrode solution at 37°C . Responses were recorded on a smoked drum by means of an isotonic lever. RESULTS
The e,~ect of the crude venom and `mammal' toxins I and II on the guinea pig ileum
The addition of each of the following substancxs, crude venom (100-2000 ng per ml), toxin I (20-1500 ng per ml) and toxin II (1-50 ng per ml) resulted in a graded initial contraction followed by a rhythmic spasmodic activity together with an increase in toms (Fig. i). The time required for the appearance of the initial contraction was dependent on the dose. When crude venom, toxin I and II were applied in doses of 700, 500 and 25 ng per ml, respectively, the initial contraction was maximal within 20-30 sec, and demonstrated a spike-like shape. Smaller doses gave the initial contraction after variable lag periods. Thus 3 ng per ml of Tx II and 100 ng per ml of crude venom resulted in an appearance of the initial contraction only after 10 and 5 min, respectively . Large repetitive doses of the above toxins and venom followed by washings, gave decreasing contractions until the ileum became completely insensitive (tachyphylaxis) (Fig. 2). Prolonged application
hka. 1. Trn; IIBAL RHSPON88 TO PROIANQ® APPLICAITON OF A. australia 'u"un,~ "* ~ roxnv I (Tl, 500 ng/ml) POLLOWBD BY A DSPHFS~D RHSPOI~B OF sBVBaAL AOON~Ia. Aoetykholine (a) 6 nglml, eemtanin (s)100 ng/ml, nicotine (n)1000 ng/ml. Time base 10 min. TOYICON t976 Yol l~
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Pte. 2. T~e ** e" * aesror>ise "ro tAxos >~srrnvs noses or d. auahnlLr 'ease.' soxn~ I. Arrowpoints to theapplication of 1"S ~g/ml of toxin I for 90 sec followed by 2 min of washing. Note the subsequent reduced response to several agoniats. Aeatylcholine (a) 6 ng/ml, histamine (h) 6 ng/ml, serotine (s) SO ng/ml, nicotine (a)1 "2 Ng/ml. Time base 2 min.
of the toxins depressed the ileal response to various agonists, especially nicotine (Figs. 1 and 2). The excitatory effect of the crude venom and the `mammal' toxins was: (1) completely inhibited by atropine and potentiated by eserine (Fig. 3), indicating an involvement of the cholinergic system, (2) not affected by Benadryl or methysergide indicating that hi . amine and seronin are not involved, (3) inhibited by tetrodotoxin and morphine, indicating a presynaptic site of action, (4) not affected by hexamethonium, ruling out a ganglionic site of action and leaving the postganglionic axon as the probable site of stimulation.
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FYo. 3. Tee ro~r~rn~aox mr sesame of ~ n~rrrxL ~L ooa~crloav n~nvcam mr .! . arcrtmlis `eue®ur.' so~m+>a I (TI, e) .earo II (T2, b). Note also decrease in lag paIod following eserine application. Aoetylcholine (Ach) 4 ngJml, Toxin I (Tl)100 ng/ml, Tmdn II (T2)10 ng/ml. Time base 2 min. TOälCON 1976 YoG !t
374
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l?Yo. 4. Tmi n C~i xespor~ z+o errllceTiox oa .! . aumoKr 'cavarsca~x' z+o~ma. (a) Prolonged application of 750 ng/ml of the toxin (Cl) followed by a depressed response to several agonists. Aeetylcholine (a) 8 ng/ml, histamine (h) 3 ng/ml, saotonin (s) 50 ng/ml, nicotine (n) 600 ng/ml. Time base 15 min. (b) The 'stepwise' deal response to small repetitive dosos of 'crustacean' toxin. Aoetylcholine (a)15 ng/ml. The arrows point to the application of Za0 ng of 'cruataoeaa' toxin per mL Time base 5 min. The e ~ect of the `crustacean' toxfn on the g:dnea pig ileum The addition of the `crustacean' toxin (0"1 to 3"0 ~g per ml) to the deal preparation resulted in a graded and slow contraction which, upon reaching a dose dependent height, remained stable over a 3 hr period (Fig. 4a). Repetitive addition of various submaximal doses without washing resulted in a step-wise increase of the deal toms, as long as the maximal contraction of the preparation was not reached (Fig . 4b). There was no appearance of rhythmic phenomena. Prolonged application of large doses of `crustacean' toxin (above 700 ng per ml) caused depression of agonist-induced contractions (Fig. 4a); in fact, nicotine was depressed almost completely in some cases (3 out of 6 experiments) . When successive supramaximal doses were applied to the preparation with washings between, the ileum became insensitive . T70X1CON 1976 YoL lI
Scorpion Toxins on Ileum
~A1r
375
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1?Ya. S. T~ ~m or waeornea (e) wrm rerao~noz+oxnv (b) ox rm3 '(atvarwe~wrt' ~roxnv n~rovc® ooxnewcrnox os ~ uses . 'Cruatactsn' toxin (CT) 750 ng/m1, atropine (Atr) ?S ng/ml, tetrodotoxin fIT}~ 50 na/ml. Time base 10 min.
Atropine, morphine and tetrodotoxin inhibited the `crustacean' toxin induced contraction of the guinea pig ileum. Application of atropine (Fig. Sa) and tetrodotoxin (Fig. Sb) to the sustained contraction returned the preparation to its normal tone . Hexamethonium, benadryl and methysergide had no effect. i?serine potentiated the response of the ileum. The e,,~`ect of the `insect' toxin on the guinea pig ileum
The `insect' toxin when applied in doses up to 1 S ~g per ml was inactive . At a concentration of 20 ~g per ml it gave only a slight and transitory increase in tone . DISCUSSION
The results with the crude venom and the `mammal' toxins I and II, suggest that their stimulatory effect follows from a postganglionic presynaptic activation, resulting is the release of a cholinergic transmitter. The stimulatory effect is characterized by an increased tonne and typical spasmodic rhythmic contraction of the ileum. The mechanism of this rhythmic phenomenon ie not fully understood . Abolition ofthe autorhythmicity by atropine and tetrodotoxin suggests the possibility that the rhythmic behaviour is an expression of a 'pulsatory' release of cholinergic transmitter, although it may also be due to some endemic muscular property of the preparation. The venom and toxins decreased the response of the preparation to several agonists. The depression of the response to nicotine and at least partially to serotonin (G+Dnust and PICARELLI, 1957) may be attributed to the depletion of the cholinergic transmitter. The decreased response to the directly stimulating smooth muscle agonists, histamine and acetylcholine, is probably due to some direct effect on the muscle. Induction of strong autorhythmic spasmodic activity by toxin II of A. australis and its suppression with atropine or tetrodotoxin as well as the involvement of a cholinergic releasing mechanism has been previously indicated by Twz~FS-D~~t$ (1972) and Twz~E-DEriSUtE and ANDxu.i.oN (1973). The present work confirms the above data contributing some new essential information : (1) the above-mentioned activities are due to postganglionic stimulation. (2) The venom and toxins also have a postsynaptic depressant effect . (3) Only a cholinergic transmitter mechanism is involved in the venom or toxin 710YICONl976 YoL l~
376
MSLVII~i TINTPULVSR, TAMAR ZSRACHIA and SLIAHU ZLOTKIN
induced activity of A. australls. (4) The `mammal' toxin I, which has only about 4 ~ of the activity of toxin II, qualitatively mimics the action of the latter . This last point is interesting, since a recent study (Cm~rMOL et al., 1974) showed qualitative differences in the cxtrdiovascular activity of these two toxins . A postganglionic and presynaptic cholinergic origin of the smooth muscle contractile activity was recently demonstrated with the toxin (tityustoxin) of the scorpion 7Ytyus serrulatus on rat ileum (Cuxx~ Mta.o et al., 197 . Comparison of the above information concerning the action of tityustoxin with that of the `m moral' toxins of A. australis indicates several differences: (1) The rhythmic contractile activity of tityustoxin was only partially blocked with atropine, suggesting the involvement of additional mediators, perhaps substance P (Cuxx~ Mr~.o et al., 1973). (2) In an atropinized preparation the tityustoxin induced response began with a sympatholytically-blockable transitory relaxation (never seen with Tx I and II of A. austrahs). The main purpose, however, of the present work was to examine the specificity of the scorpion toxins . From this point of view the `insect' toxin should be considered inactive . In huge doses up to 20 ~g per ml it had almost no effect on the ileum [20 I~g are equivalent to approximately 7 mg of crude venom (Zto~r~t et al., 1971)] . The `crustacean' toxin on the other hand, clearly demonstrated an excitatory effect on the deal preparation. Its action was : (1) completely inhibited by atropine, (2) potentiated by eserine, (3) tachyphylactic, (4) blocked by tetrodotoxin and morphine and (5) unaffected by hexamethonium. Therefore, its excitatory effect follows from a postganglionic presynaptic action releasing a cholinergic transmitter, thus mimicking the action of the crude venom and its mammal toxins. The postsynaptic effects expressed in a reduced response to agonists, as indicated for the `mammal' toxins were equally demonstrated by the `crustacean' toxin. However, essential differences were evident. (1) The initial contraction induced by the `crustacean' toxin (even by high doses) was slower than that induced by the `mammal' toxins . (2) In contrast to `mammal' toxins, the `crustacean' toxin exhibits no rhythmically spasmodic behaviour. (3) Repetitive additions of `crustacean' toxin without washing resulted in additive elevations of the tonus, up to the maximal response of the preparation. This is in contrast to the effect of the `mammal' toxins, which regardless of the dose eventually gave maximal or near-maximal contractions . The `crustacean' toxin induced sustained elevations in tone appear to be due to the releasing behaviour of the cholinergic transmitter, since it is blocked by atropine, morphine and tetrodotoxin . Thus, the `crustacean' toxin may induce a sustained continuous transmitter release in comparison to the hypothetical `pulsatory' release of transmitter induced by the `mammal' toxins . The most significant point, however, is the ability of the sculled `crustacean' toxin to affect a mammalian neuromuscular system, via essentially the same mechanism as that of the `mammal' toxins . Most recently the above toxins of A. australis were tested on nerve muscle preparations of an insect (Wu.~e et al., in press) crustacean (RATIiMAYSR et al., in press) and arachnid (RUFIIAND et al., in preparation) . It has been found that the `crustacean' toxin, which was the most potent in inducing excitatory effects in the crustacean as well as arachnid preparations was also active in the insect preparation. The `mammal' toxin I which in the present work was found to be much less active than toxin II, was definitely more potent than the latter in affecting the neuromuscular systems of the arthropods . The `insect' toxin which was inactive in the present preparation, was equally inactive in the crustacean as well as arachnid preparations, but was the most potent in affecting the insect neuromuscular preparation, due to an excitatory presynaptic effect on TO%ICON1976 YoL I~
Searploa Toxine on Ileum
377
the motor nerve. As such it may bo considered as a highly selective compound and valuable pharmacological tool . Acknowledgenent~-The authors are aratd'ul to ProL Mnewrrow sect Pmf. RocawT, Facultd de Mbdeclne, Secteur Nord, Biochimie, lVlatawlle, for their bad donation of the 'mammal' toxins I sad II. REFERENCB.S n'Alsia o, V., Zt o~rw, E., Mrnwrmw, F., L~1Z[Y, S. and BsTrlru, S. (1972). The effect of scorpion venom and pure toxic on the cockroach central nervous system. Toxkon 10, 399. Howetwx, W. C., Rwrm, M. J. and WFar, Ci. H. (1972) Textbook of Pharmacology, 8 pp. Oxford : Hlackwell. Bv~an~o, E., Bawnnva, A. F., Joxes, A. W. and To~rw, T., (Eds.), (1970) Snwoth Mrcsck. London : Arnold . Cm;r~ot., J., Hovxn,~, F., Rocs-ARV~e, M. and HOB, J. (1974) Action cardiovascailaire de trois venins de scorpions nord afrkaina (Leturua qutnqueatrlatua, Balboa occita~wa et Androctonua mratralfa) et de deux toxines extraites de ßm d'entro eux. Toxkon lT., 241. Caw M~.o, J. R, F~e~Mwiw, L., TwrvxT, W. L. and Mwarw, T. A. (1973) Mecbaniam of action of purified scorpion toxin an the isolated rat iateatine. Toxtcon 11, 81 . Fxxnes-Mwrw, L. and Duaz, C. R (1970) Pharmacological action of a purified scorpion torirr in the rat . Toxkon 8, 132. (3wnnvM, J. H. and Picwnsui, Z. P. (1957) Two kinds of tryptamine receptor . Br. J. Pharnrac. Clunwther. 29, 323. Mmwxnw, F., RocawT, H. and LasanzsY, S. (1964) Sur lea neurotoxiaea de deux espèces de scorpions nord africains. I. Purification de neurotoxina (scorpamines) d'Androetonua austrnlia (L.) et de &ttluu occltanua (Am.) . Toxlavn 2, Sl . Mmwxnw, F., Kvrs~rwx, C., RocawT, H., RocawT, C. and Lia~TZYY, S. (1970) Purification of animal neurotoxins. Isolation and characteri~tion of eleven n~rotorina from the venom of the scorpions Androctonus auatnalLs Hector, But/wa occltanus trmetanua and Letrvua grdrtqueatriatua grdngneatriatua. Flu. J. Blochent. 16, 514. Pwxs~, M. C., Miaraoiu-NwTwuzr, Cl . and Bsrtn~¢, 3. (1973) Effect of scorpion venom and its fractions on the crayfish stretch receptor organ. Toxtoon 11, 283. Pw~esox, R A. (1962) Pharmacological action of scorpion venom on intestinal smooth muscle . Toxic. appl. Plearnmc. 4, 710. Iüa~wYSa, W., WALTHSR, Cme. and ZLOrxn~t, E. (in press) Tho effect of different toxins from scorpion venom on neuromuscular transmission and nerve action potentials in the crayfish. J. comp. Blochan. Phyatol. RocawT, C., ROCHAT, H., Mnswrmw, F. and I~zeY, S. (1967) Purification and some properties of the neurotoxins of AndmctonWa arrstralla Hector . Biochemistry 6, 578. RocawT, H., RocxwT, C., Kors~rwx, C., Mmwxnw, F., Id8~1ZYY, S. and EDMAN, P. (1970) Scorpion neurotoxins : a family of homologous proteins . FEBS Lett .10, 359. Rvr~xn, M., ZLO~rsw, E. and RATEIIIlAYSR, W. (in preparation) The effect of different scorpion toxins on a spider nerve~uacle preparation. Twz~+B-D~exs, F. (1972) VeOin de scorpion, calcium et emission d'aoEtylcholiae par lea fibres nerveuses dans fildon de cobaye. C. r. hebd. Saar~c. Atwd. Sci., Parla Saiea D 275, 3021 . Twz~+rerDBP~, F. and ArmxII.wx, P. (1973) Secretion d'ao6tylcholine provoqude par le via de scorpion dear filéoa de cobaye et sa suppression par )a tétrodotoxino. C. r. leebd . Seanc. Read. Sd ., Parla S~iea D 276,1631 . Wwr.~, Cax., Zr o~mx, E. and RATHINAYER, W. (inpress) The action of different toxins from the scorpion Androctomua auatralla Hector on a locust nerve muscle preparation . J. Insect Phystol. Z~~r, E., RoeawT, H., Kvrerwx, C., Mmwrmw, F. and L~errcs:Y, S. (1971) Puri~ation and properties of the "insact~ toria from the venom of thescorpion Androctonua auatraliaHector . Blochbrrie 53,1073. Ztoz~r, E., MIIewrmw, F. and L~szzxx, S. (1972a) Pmteina toric to mammals end insects in six scorpion venons. Toxkon 10, 207. Ztars~, E., Mmwrrow, F. and LLlATZYY, S. (1972b) A toxic factor to crustacean is the venom of the scorpion Androctomra auatmlfa Hector. Toxtoon 10, 211. Ztacw, E., Mwazn~z, C3., Roc~wr, H. and Mmwrrow, F. (197 A protda toric to cruataoea from the venom of the scorpion Androctomu arratmlta Hector. Insect Btoclunr. S, 1073 .
T~OXICON 1976 Yol. If
vr. snan. t~s mon r~...
~ clreu s~n m.
THE ACTION OF TOXINS DERIVED FROM SCORPION VENOM ON THE ILEAL SMOOTH MUSCLE PREPARATION* MEC,vua T1NTPU1,vEx,t Tax Z>~uct~ and F.~.uHV ZtoTglrt Department of Entomology and Venomous Anfmals, The Hebrew University of Jerusalem, Jerusalem, Israel (Accepted for prrbltrntlon
9
March
197
M. Tnvlruc vBa, T . Z8RAC8IA and B. Zwr~r. The action of toxins derived from scorpion venom on the deal smooth muscle preparation. Toxicon 14, 371-377,1976.-The'inse~t','cxuatacean' and `mammal' I end II toxins previously purified from tho venom of the scorpion Androcttmrct arratm/Lv Hector according to their fiy larvae paralysis, isopoda paralysis and mice lethality, r+espectlvely, were applied on the guinea pig ilaim smooth muscle preparation and it has bees found that : (1) tho 'crustacean' toxin induced a sustained-prolonged contracdon is contrast to the rhythmic spasmodic deal behaviour caused by the crude venom and the `mammal' toxins. (2) The excitatory effect of these substances wan due to a postganglionic ptraynaptic stimulation rosultin8 in the release of a cholinergic transmitter. (3) Prolonged application ofthe 'mammal' as well as `crustacean' toxins, caused e depression ofpostsynaptic reactivity as observed in a reduced deal response to several agonists. (4) The 'insect' toxin, wan inactive in the t7eum smooth musde preparation. INTRODUCTION Wirx T~ aid of simple bioassays, based on lethality or paralysis of test animals, it has been shown that the activity of scorpion venom on arthropods is due to specific factors (Ztarx~ et al., 1971, 1972a, b), that differ from the well-known toxins active on mammals (MIRANDA et al., 1964, 1970; ROCx~.T et al., 1967, 1970) . Furthermore, the paralysis and death of isopods (Crustacea) by A. australis venom is due to a protein factor different from the one causing paralysis and death of insects (ZLOTSIN et al., 1972b, 1975) . The small dosage, speed of paralysis and the symptomatology indicate that these effects are probably due to a neurotoxic action of these specific factors. These materials have also been tested in a more specific fashion : The `insect' toxin (ZLO~rxua et al., 1971) blocked synaptic transmission in the sixth abdominal ganglion of the central nervous system of Periplaneta amerlcana (n'Arns.to et al., 1972). The most potent `mammal' toxin (Tx In isolated from the venom of the same scorpion (MIRANDA et al., 1970) was inactive. Likewise, the `crustacean' toxin fraction induced an excitatory blockade of the crayfish stretch receptor (PANSA et al., 1973) while Tx II and the `.insect' toxin were ineffective. Thus, it has been assumed that the specificity of the above toxins is based on their specific affinity for neural systems of the relevant groups of organisms . It seemed pertinent to perform an examination of the arthropod toxins by applying them to a vertebrate system. The smooth muscle preparation of the guinea pig ileum has 'Supported by orant Nb 730, from the U.S.-Israel Binational Scieaoe Foundation. tPtesent address : Queens University, Dept. Biology, Kingston, Canada K7L 3N6. 371 710XICON 1976 Yol. I ~
372
MELVII~i TiNTPULVER, TAMAR ZERACHIA aad Ei .iAHÜ ZhOTKiN
been used to test the action of scorpion venoms and toxins (PATr~sox,1962 ; FRaut>ß-NL~tA and Du~iz, 1970 ; CuxtiA MEio et al., 1973), thus supplying a basis for comparison with our present studies. MA~~ Q AND METHODS Crude venom from Androctonur xuatralla Hector was electrically milked from the ecorpiona and subsequenUy lyophilized. The 'mammal' toxins I and II, `insect toxin' (iT1, and 'crustacean' toxin (CT) were purißed from the same venom (M~~~ et x1 .,1970 ; Ztar~ et x1.,1971,1976) . The following drugs wore used : acetyk:holine chloride, atropine sulphate, physostigmine (eserine) sulphate, diplrenhydtamino hydrochloride (Benadryl), hexamethonium bromide, S-hydroxy-tryptamiae creatinine sulphate (saotonin), nicotine sulphate and histamine dihydrochloride, all obtained from Sigma Co . ; morphine sulphate (Merck Co.) ; methyaergide maleate (Deaeril) (Saadoz Co.) ; tetrodotoxin citrate (Sanlgo--C~lbiocheai). All chemicals wero of analytical grade. The appropriate Tyrode solution was composed of (g per 1) : NaC18~0; KC10~2 ; MgC12 0-0S ; CaClz 0"2; NaH2P04 005; NaHC031-0 and glucose 1-0 (Howeux et x1.,1972). Starved male guinea Pigs were killed by a blow an the head. The ileum was removed, washed and segmenta of 2-3 cm were sectioned and susp~ded in a 15 ml organ bath containing seratod Tyrode solution at 37°C . Responses were recorded on a smoked drum by means of an isotonic lever. RESULTS
The e,~ect of the crude venom and `mammal' toxins I and II on the guinea pig ileum
The addition of each of the following substancxs, crude venom (100-2000 ng per ml), toxin I (20-1500 ng per ml) and toxin II (1-50 ng per ml) resulted in a graded initial contraction followed by a rhythmic spasmodic activity together with an increase in toms (Fig. i). The time required for the appearance of the initial contraction was dependent on the dose. When crude venom, toxin I and II were applied in doses of 700, 500 and 25 ng per ml, respectively, the initial contraction was maximal within 20-30 sec, and demonstrated a spike-like shape. Smaller doses gave the initial contraction after variable lag periods. Thus 3 ng per ml of Tx II and 100 ng per ml of crude venom resulted in an appearance of the initial contraction only after 10 and 5 min, respectively . Large repetitive doses of the above toxins and venom followed by washings, gave decreasing contractions until the ileum became completely insensitive (tachyphylaxis) (Fig. 2). Prolonged application
hka. 1. Trn; IIBAL RHSPON88 TO PROIANQ® APPLICAITON OF A. australia 'u"un,~ "* ~ roxnv I (Tl, 500 ng/ml) POLLOWBD BY A DSPHFS~D RHSPOI~B OF sBVBaAL AOON~Ia. Aoetykholine (a) 6 nglml, eemtanin (s)100 ng/ml, nicotine (n)1000 ng/ml. Time base 10 min. TOYICON t976 Yol l~
J~JJ
Pte. 2. T~e ** e" * aesror>ise "ro tAxos >~srrnvs noses or d. auahnlLr 'ease.' soxn~ I. Arrowpoints to theapplication of 1"S ~g/ml of toxin I for 90 sec followed by 2 min of washing. Note the subsequent reduced response to several agoniats. Aeatylcholine (a) 6 ng/ml, histamine (h) 6 ng/ml, serotine (s) SO ng/ml, nicotine (a)1 "2 Ng/ml. Time base 2 min.
of the toxins depressed the ileal response to various agonists, especially nicotine (Figs. 1 and 2). The excitatory effect of the crude venom and the `mammal' toxins was: (1) completely inhibited by atropine and potentiated by eserine (Fig. 3), indicating an involvement of the cholinergic system, (2) not affected by Benadryl or methysergide indicating that hi . amine and seronin are not involved, (3) inhibited by tetrodotoxin and morphine, indicating a presynaptic site of action, (4) not affected by hexamethonium, ruling out a ganglionic site of action and leaving the postganglionic axon as the probable site of stimulation.
Ach TI
Ach
E
TI
Ach T2
Ach
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FYo. 3. Tee ro~r~rn~aox mr sesame of ~ n~rrrxL ~L ooa~crloav n~nvcam mr .! . arcrtmlis `eue®ur.' so~m+>a I (TI, e) .earo II (T2, b). Note also decrease in lag paIod following eserine application. Aoetylcholine (Ach) 4 ngJml, Toxin I (Tl)100 ng/ml, Tmdn II (T2)10 ng/ml. Time base 2 min. TOälCON 1976 YoG !t
374
MSLVIIV TIIVTPULVSR, TAMAR ZBRACHIA sad BLiAiiU ZLOTKIIv
~naha GT
nsho
l?Yo. 4. Tmi n C~i xespor~ z+o errllceTiox oa .! . aumoKr 'cavarsca~x' z+o~ma. (a) Prolonged application of 750 ng/ml of the toxin (Cl) followed by a depressed response to several agonists. Aeetylcholine (a) 8 ng/ml, histamine (h) 3 ng/ml, saotonin (s) 50 ng/ml, nicotine (n) 600 ng/ml. Time base 15 min. (b) The 'stepwise' deal response to small repetitive dosos of 'crustacean' toxin. Aoetylcholine (a)15 ng/ml. The arrows point to the application of Za0 ng of 'cruataoeaa' toxin per mL Time base 5 min. The e ~ect of the `crustacean' toxfn on the g:dnea pig ileum The addition of the `crustacean' toxin (0"1 to 3"0 ~g per ml) to the deal preparation resulted in a graded and slow contraction which, upon reaching a dose dependent height, remained stable over a 3 hr period (Fig. 4a). Repetitive addition of various submaximal doses without washing resulted in a step-wise increase of the deal toms, as long as the maximal contraction of the preparation was not reached (Fig . 4b). There was no appearance of rhythmic phenomena. Prolonged application of large doses of `crustacean' toxin (above 700 ng per ml) caused depression of agonist-induced contractions (Fig. 4a); in fact, nicotine was depressed almost completely in some cases (3 out of 6 experiments) . When successive supramaximal doses were applied to the preparation with washings between, the ileum became insensitive . T70X1CON 1976 YoL lI
Scorpion Toxins on Ileum
~A1r
375
~ TTX
1?Ya. S. T~ ~m or waeornea (e) wrm rerao~noz+oxnv (b) ox rm3 '(atvarwe~wrt' ~roxnv n~rovc® ooxnewcrnox os ~ uses . 'Cruatactsn' toxin (CT) 750 ng/m1, atropine (Atr) ?S ng/ml, tetrodotoxin fIT}~ 50 na/ml. Time base 10 min.
Atropine, morphine and tetrodotoxin inhibited the `crustacean' toxin induced contraction of the guinea pig ileum. Application of atropine (Fig. Sa) and tetrodotoxin (Fig. Sb) to the sustained contraction returned the preparation to its normal tone . Hexamethonium, benadryl and methysergide had no effect. i?serine potentiated the response of the ileum. The e,,~`ect of the `insect' toxin on the guinea pig ileum
The `insect' toxin when applied in doses up to 1 S ~g per ml was inactive . At a concentration of 20 ~g per ml it gave only a slight and transitory increase in tone . DISCUSSION
The results with the crude venom and the `mammal' toxins I and II, suggest that their stimulatory effect follows from a postganglionic presynaptic activation, resulting is the release of a cholinergic transmitter. The stimulatory effect is characterized by an increased tonne and typical spasmodic rhythmic contraction of the ileum. The mechanism of this rhythmic phenomenon ie not fully understood . Abolition ofthe autorhythmicity by atropine and tetrodotoxin suggests the possibility that the rhythmic behaviour is an expression of a 'pulsatory' release of cholinergic transmitter, although it may also be due to some endemic muscular property of the preparation. The venom and toxins decreased the response of the preparation to several agonists. The depression of the response to nicotine and at least partially to serotonin (G+Dnust and PICARELLI, 1957) may be attributed to the depletion of the cholinergic transmitter. The decreased response to the directly stimulating smooth muscle agonists, histamine and acetylcholine, is probably due to some direct effect on the muscle. Induction of strong autorhythmic spasmodic activity by toxin II of A. australis and its suppression with atropine or tetrodotoxin as well as the involvement of a cholinergic releasing mechanism has been previously indicated by Twz~FS-D~~t$ (1972) and Twz~E-DEriSUtE and ANDxu.i.oN (1973). The present work confirms the above data contributing some new essential information : (1) the above-mentioned activities are due to postganglionic stimulation. (2) The venom and toxins also have a postsynaptic depressant effect . (3) Only a cholinergic transmitter mechanism is involved in the venom or toxin 710YICONl976 YoL l~
376
MSLVII~i TINTPULVSR, TAMAR ZSRACHIA and SLIAHU ZLOTKIN
induced activity of A. australls. (4) The `mammal' toxin I, which has only about 4 ~ of the activity of toxin II, qualitatively mimics the action of the latter . This last point is interesting, since a recent study (Cm~rMOL et al., 1974) showed qualitative differences in the cxtrdiovascular activity of these two toxins . A postganglionic and presynaptic cholinergic origin of the smooth muscle contractile activity was recently demonstrated with the toxin (tityustoxin) of the scorpion 7Ytyus serrulatus on rat ileum (Cuxx~ Mta.o et al., 197 . Comparison of the above information concerning the action of tityustoxin with that of the `m moral' toxins of A. australis indicates several differences: (1) The rhythmic contractile activity of tityustoxin was only partially blocked with atropine, suggesting the involvement of additional mediators, perhaps substance P (Cuxx~ Mr~.o et al., 1973). (2) In an atropinized preparation the tityustoxin induced response began with a sympatholytically-blockable transitory relaxation (never seen with Tx I and II of A. austrahs). The main purpose, however, of the present work was to examine the specificity of the scorpion toxins . From this point of view the `insect' toxin should be considered inactive . In huge doses up to 20 ~g per ml it had almost no effect on the ileum [20 I~g are equivalent to approximately 7 mg of crude venom (Zto~r~t et al., 1971)] . The `crustacean' toxin on the other hand, clearly demonstrated an excitatory effect on the deal preparation. Its action was : (1) completely inhibited by atropine, (2) potentiated by eserine, (3) tachyphylactic, (4) blocked by tetrodotoxin and morphine and (5) unaffected by hexamethonium. Therefore, its excitatory effect follows from a postganglionic presynaptic action releasing a cholinergic transmitter, thus mimicking the action of the crude venom and its mammal toxins. The postsynaptic effects expressed in a reduced response to agonists, as indicated for the `mammal' toxins were equally demonstrated by the `crustacean' toxin. However, essential differences were evident. (1) The initial contraction induced by the `crustacean' toxin (even by high doses) was slower than that induced by the `mammal' toxins . (2) In contrast to `mammal' toxins, the `crustacean' toxin exhibits no rhythmically spasmodic behaviour. (3) Repetitive additions of `crustacean' toxin without washing resulted in additive elevations of the tonus, up to the maximal response of the preparation. This is in contrast to the effect of the `mammal' toxins, which regardless of the dose eventually gave maximal or near-maximal contractions . The `crustacean' toxin induced sustained elevations in tone appear to be due to the releasing behaviour of the cholinergic transmitter, since it is blocked by atropine, morphine and tetrodotoxin . Thus, the `crustacean' toxin may induce a sustained continuous transmitter release in comparison to the hypothetical `pulsatory' release of transmitter induced by the `mammal' toxins . The most significant point, however, is the ability of the sculled `crustacean' toxin to affect a mammalian neuromuscular system, via essentially the same mechanism as that of the `mammal' toxins . Most recently the above toxins of A. australis were tested on nerve muscle preparations of an insect (Wu.~e et al., in press) crustacean (RATIiMAYSR et al., in press) and arachnid (RUFIIAND et al., in preparation) . It has been found that the `crustacean' toxin, which was the most potent in inducing excitatory effects in the crustacean as well as arachnid preparations was also active in the insect preparation. The `mammal' toxin I which in the present work was found to be much less active than toxin II, was definitely more potent than the latter in affecting the neuromuscular systems of the arthropods . The `insect' toxin which was inactive in the present preparation, was equally inactive in the crustacean as well as arachnid preparations, but was the most potent in affecting the insect neuromuscular preparation, due to an excitatory presynaptic effect on TO%ICON1976 YoL I~
Searploa Toxine on Ileum
377
the motor nerve. As such it may bo considered as a highly selective compound and valuable pharmacological tool . Acknowledgenent~-The authors are aratd'ul to ProL Mnewrrow sect Pmf. RocawT, Facultd de Mbdeclne, Secteur Nord, Biochimie, lVlatawlle, for their bad donation of the 'mammal' toxins I sad II. REFERENCB.S n'Alsia o, V., Zt o~rw, E., Mrnwrmw, F., L~1Z[Y, S. and BsTrlru, S. (1972). The effect of scorpion venom and pure toxic on the cockroach central nervous system. Toxkon 10, 399. Howetwx, W. C., Rwrm, M. J. and WFar, Ci. H. (1972) Textbook of Pharmacology, 8 pp. Oxford : Hlackwell. Bv~an~o, E., Bawnnva, A. F., Joxes, A. W. and To~rw, T., (Eds.), (1970) Snwoth Mrcsck. London : Arnold . Cm;r~ot., J., Hovxn,~, F., Rocs-ARV~e, M. and HOB, J. (1974) Action cardiovascailaire de trois venins de scorpions nord afrkaina (Leturua qutnqueatrlatua, Balboa occita~wa et Androctonua mratralfa) et de deux toxines extraites de ßm d'entro eux. Toxkon lT., 241. Caw M~.o, J. R, F~e~Mwiw, L., TwrvxT, W. L. and Mwarw, T. A. (1973) Mecbaniam of action of purified scorpion toxin an the isolated rat iateatine. Toxtcon 11, 81 . Fxxnes-Mwrw, L. and Duaz, C. R (1970) Pharmacological action of a purified scorpion torirr in the rat . Toxkon 8, 132. (3wnnvM, J. H. and Picwnsui, Z. P. (1957) Two kinds of tryptamine receptor . Br. J. Pharnrac. Clunwther. 29, 323. Mmwxnw, F., RocawT, H. and LasanzsY, S. (1964) Sur lea neurotoxiaea de deux espèces de scorpions nord africains. I. Purification de neurotoxina (scorpamines) d'Androetonua austrnlia (L.) et de &ttluu occltanua (Am.) . Toxlavn 2, Sl . Mmwxnw, F., Kvrs~rwx, C., RocawT, H., RocawT, C. and Lia~TZYY, S. (1970) Purification of animal neurotoxins. Isolation and characteri~tion of eleven n~rotorina from the venom of the scorpions Androctonus auatnalLs Hector, But/wa occltanus trmetanua and Letrvua grdrtqueatriatua grdngneatriatua. Flu. J. Blochent. 16, 514. Pwxs~, M. C., Miaraoiu-NwTwuzr, Cl . and Bsrtn~¢, 3. (1973) Effect of scorpion venom and its fractions on the crayfish stretch receptor organ. Toxtoon 11, 283. Pw~esox, R A. (1962) Pharmacological action of scorpion venom on intestinal smooth muscle . Toxic. appl. Plearnmc. 4, 710. Iüa~wYSa, W., WALTHSR, Cme. and ZLOrxn~t, E. (in press) Tho effect of different toxins from scorpion venom on neuromuscular transmission and nerve action potentials in the crayfish. J. comp. Blochan. Phyatol. RocawT, C., ROCHAT, H., Mnswrmw, F. and I~zeY, S. (1967) Purification and some properties of the neurotoxins of AndmctonWa arrstralla Hector . Biochemistry 6, 578. RocawT, H., RocxwT, C., Kors~rwx, C., Mmwxnw, F., Id8~1ZYY, S. and EDMAN, P. (1970) Scorpion neurotoxins : a family of homologous proteins . FEBS Lett .10, 359. Rvr~xn, M., ZLO~rsw, E. and RATEIIIlAYSR, W. (in preparation) The effect of different scorpion toxins on a spider nerve~uacle preparation. Twz~+B-D~exs, F. (1972) VeOin de scorpion, calcium et emission d'aoEtylcholiae par lea fibres nerveuses dans fildon de cobaye. C. r. hebd. Saar~c. Atwd. Sci., Parla Saiea D 275, 3021 . Twz~+rerDBP~, F. and ArmxII.wx, P. (1973) Secretion d'ao6tylcholine provoqude par le via de scorpion dear filéoa de cobaye et sa suppression par )a tétrodotoxino. C. r. leebd . Seanc. Read. Sd ., Parla S~iea D 276,1631 . Wwr.~, Cax., Zr o~mx, E. and RATHINAYER, W. (inpress) The action of different toxins from the scorpion Androctomua auatralla Hector on a locust nerve muscle preparation . J. Insect Phystol. Z~~r, E., RoeawT, H., Kvrerwx, C., Mmwrmw, F. and L~errcs:Y, S. (1971) Puri~ation and properties of the "insact~ toria from the venom of thescorpion Androctonua auatraliaHector . Blochbrrie 53,1073. Ztoz~r, E., MIIewrmw, F. and L~szzxx, S. (1972a) Pmteina toric to mammals end insects in six scorpion venons. Toxkon 10, 207. Ztars~, E., Mmwrrow, F. and LLlATZYY, S. (1972b) A toxic factor to crustacean is the venom of the scorpion Androctomra auatmlfa Hector. Toxtoon 10, 211. Ztacw, E., Mwazn~z, C3., Roc~wr, H. and Mmwrrow, F. (197 A protda toric to cruataoea from the venom of the scorpion Androctomu arratmlta Hector. Insect Btoclunr. S, 1073 .
T~OXICON 1976 Yol. If